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In an announcement televised on C-Span, President Elect Joseph Biden announced his new Science Team to advise on science policy matters, as part of the White House Advisory Committee on Science and Technology. Below is a video clip and the transcript, also available at
there have been many instances of off-target effects where genes, other than the selected target, are edited out. This ‘off-target’ issue has hampered much of the utility of CRISPR in gene-therapy and CART therapy
However, an article in Science by Jon Cohen explains a Nature paper’s finding of a new tool in the CRISPR arsenal called prime editing, meant to increase CRISPR specificity and precision editing capabilities.
Primeediting promises to be a cut above CRISPR Jon Cohen CRISPR, an extraordinarily powerful genome-editing tool invented in 2012, can still be clumsy. … Primeediting steers around shortcomings of both techniques by heavily modifying the Cas9 protein and the guide RNA. … ” Primeediting “well may become the way that disease-causing mutations are repaired,” he says.
The effort, led by Drs. David Liu and Andrew Anzalone at the Broad Institute (Cambridge, MA), relies on the modification of the Cas9 protein and guide RNA, so that there is only a nick in a single strand of the double helix. The canonical Cas9 cuts both strands of DNA, and so relies on an efficient gap repair activity of the cell. The second part, a new type of guide RNA called a pegRNA, contains an RNA template for a new DNA sequence to be added at the target location. This pegRNA-directed synthesis of the new template requires the attachment of a reverse transcriptase enzymes to the Cas9. So far Liu and his colleagues have tested the technology on over 175 human and rodent cell lines with great success. In addition, they had also corrected mutations which cause Tay Sachs disease, which previous CRISPR systems could not do. Liu claims that this technology could correct over 89% of pathogenic variants in human diseases.
A company Prime Medicine has been formed out of this effort.
As was announced, prime editing for human therapeutics will be jointly developed by both Prime Medicine and Beam Therapeutics, each focusing on different types of edits and distinct disease targets, which will help avoid redundancy and allow us to cover more disease territory overall. The companies will also share knowledge in prime editing as well as in accompanying technologies, such as delivery and manufacturing.
Reader of StatNews.: Can you please compare the pros and cons of prime editing versus base editing?
The first difference between base editing and prime editing is that base editing has been widely used for the past 3 1/2 years in organisms ranging from bacteria to plants to mice to primates. Addgene tells me that the DNA blueprints for base editors from our laboratory have been distributed more than 7,500 times to more than 1,000 researchers around the world, and more than 100 research papers from many different laboratories have been published using base editors to achieve desired gene edits for a wide variety of applications. While we are very excited about prime editing, it’s brand-new and there has only been one paper published thus far. So there’s much to do before we can know if prime editing will prove to be as general and robust as base editing has proven to be.
We directly compared prime editors and base editors in our study, and found that current base editors can offer higher editing efficiency and fewer indel byproducts than prime editors, while prime editors offer more targeting flexibility and greater editing precision. So when the desired edit is a transition point mutation (C to T, T to C, A to G, or G to A), and the target base is well-positioned for base editing (that is, a PAM sequence exists approximately 15 bases from the target site), then base editing can result in higher editing efficiencies and fewer byproducts. When the target base is not well-positioned for base editing, or when other “bystander” C or A bases are nearby that must not be edited, then prime editing offers major advantages since it does not require a precisely positioned PAM sequence and is a true “search-and-replace” editing capability, with no possibility of unwanted bystander editing at neighboring bases.
Of course, for classes of mutations other than the four types of point mutations that base editors can make, such as insertions, deletions, and the eight other kinds of point mutations, to our knowledge prime editing is currently the only approach that can make these mutations in human cells without requiring double-stranded DNA cuts or separate DNA templates.
Nucleases (such as the zinc-finger nucleases, TALE nucleases, and the original CRISPR-Cas9), base editors, and prime editors each have complementary strengths and weaknesses, just as scissors, pencils, and word processors each have unique and useful roles. All three classes of editing agents already have or will have roles in basic research and in applications such as human therapeutics and agriculture.
Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2,3,4,5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay–Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.
From Anzolone et al. Nature 2019 Figure 1.
Prime editing strategy
Cas9 targets DNA using a guide RNA containing a spacer sequence that hybridizes to the target DNA site. We envisioned the generation of guide RNAs that both specify the DNA target and contain new genetic information that replaces target DNA nucleotides. To transfer information from these engineered guide RNAs to target DNA, we proposed that genomic DNA, nicked at the target site to expose a 3′-hydroxyl group, could be used to prime the reverse transcription of an edit-encoding extension on the engineered guide RNA (the pegRNA) directly into the target site (Fig. 1b, c, Supplementary Discussion).
These initial steps result in a branched intermediate with two redundant single-stranded DNA flaps: a 5′ flap that contains the unedited DNA sequence and a 3′ flap that contains the edited sequence copied from the pegRNA (Fig. 1c). Although hybridization of the perfectly complementary 5′ flap to the unedited strand is likely to be thermodynamically favoured, 5′ flaps are the preferred substrate for structure-specific endonucleases such as FEN122, which excises 5′ flaps generated during lagging-strand DNA synthesis and long-patch base excision repair. The redundant unedited DNA may also be removed by 5′ exonucleases such as EXO123.
The authors reasoned that preferential 5′ flap excision and 3′ flap ligation could drive the incorporation of the edited DNA strand, creating heteroduplex DNA containing one edited strand and one unedited strand (Fig. 1c).
DNA repair to resolve the heteroduplex by copying the information in the edited strand to the complementary strand would permanently install the edit (Fig. 1c).
They had hypothesized that nicking the non-edited DNA strand might bias DNA repair to preferentially replace the non-edited strand.
Results
The authors evaluated the eukaryotic cell DNA repair outcomes of 3′ flaps produced by pegRNA-programmed reverse transcription in vitro, and performed in vitro prime editing on reporter plasmids, then transformed the reaction products into yeast cells (Extended Data Fig. 2).
Reporter plasmids encoding EGFP and mCherry separated by a linker containing an in-frame stop codon, +1 frameshift, or −1 frameshift were constructed and when plasmids were edited in vitro with Cas9 nickase, RT, and 3′-extended pegRNAs encoding a transversion that corrects the premature stop codon, 37% of yeast transformants expressed both GFP and mCherry (Fig. 1f, Extended Data Fig. 2).
They fused a variant of M—MLV-RT (reverse transcriptase) to Cas9 with an extended linker and this M-MLV RT fused to the C terminus of Cas9(H840A) nickase was designated as PE1. This strategy allowed the authors to generate a cell line containing all the required components of the primer editing system. They constructed 19 variants of PE1 containing a variety of RT mutations to evaluate their editing efficiency in human cells
Generated a pentamutant RT incorporated into PE1 (Cas9(H840A)–M-MLV RT(D200N/L603W/T330P/T306K/W313F)) is hereafter referred to as prime editor 2 (PE2). These were more thermostable versions of RT with higher efficiency.
Optimized the guide (pegRNA) using a series of permutations and recommend starting with about 10–16 nt and testing shorter and longer RT templates during pegRNA optimization.
In the previous attempts (PE1 and PE2 systems), mismatch repair resolves the heteroduplex to give either edited or non-edited products. So they next developed an optimal editing system (PE3) to produce optimal nickase activity and found nicks positioned 3′ of the edit about 40–90 bp from the pegRNA-induced nick generally increased editing efficiency (averaging 41%) without excess indel formation (6.8% average indels for the sgRNA with the highest editing efficiency) (Fig. 3b).
The cell line used to finalize and validate the system was predominantly HEK293T immortalized cell line
Together, their findings establish that PE3 systems improve editing efficiencies about threefold compared with PE2, albeit with a higher range of indels than PE2. When it is possible to nick the non-edited strand with an sgRNA that requires editing before nicking, the PE3b system offers PE3-like editing levels while greatly reducing indel formation.
Off Target Effects: Strikingly, PE3 or PE2 with the same 16 pegRNAs containing these four target spacers resulted in detectable off-target editing at only 3 out of 16 off-target sites, with only 1 of 16 showing an off-target editing efficiency of 1% or more (Extended Data Fig. 6h). Average off-target prime editing for pegRNAs targeting HEK3, HEK4, EMX1, and FANCFat the top four known Cas9 off-target sites for each protospacer was <0.1%, <2.2 ± 5.2%, <0.1%, and <0.13 ± 0.11%, respectively (Extended Data Fig. 6h).
The PE3 system was very efficient at editing the most common mutation that causes Tay-Sachs disease, a 4-bp insertion in HEXA(HEXA1278+TATC).
References
Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 44, D862–D868 (2016).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science337, 816–821 (2012).
Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Biotechnol. 36, 765–771 (2018).
When it comes to stem cells, too much of a good thing isn’t wonderful: Producing too many new stem cells may lead to cancer; producing too few inhibits the repair and maintenance of the body.
In a paper published in Stem Cell Reports, USC researcher In Kyoung Mah, who works in the lab of Francesca Mariani, and colleagues at the University of California, San Diego, describe a key gene that maintains this critical balance. Called Prkci, the gene influences whether stem cells self-renew to produce more stem cells or differentiate into more specialized cell types, such as blood or nerves.
In their experiments, the team grew mouse embryonic stem cells, which lacked Prkci, into embryo-like structures in the lab. Without Prkci, the stem cells favored self-renewal, generating large numbers of stem cells and, subsequently, an abundance of secondary structures.
Upon closer inspection, the stem cells lacking Prkci had many activated genes typical of stem cells, and some activated genes typical of neural, cardiac and blood-forming cells. Therefore, the loss of Prkci can also encourage stem cells to differentiate into the progenitor cells that form neurons, heart muscle and blood.
Follow the pathway
Prkci achieves these effects by activating or deactivating a well-known group of interacting genes that are part of the “Notch signaling pathway.” In the absence of Prkci, the Notch pathway produces a protein that signals to stem cells to make more stem cells. In the presence of Prkci, the Notch pathway remains silent, and stem cells differentiate into specific cell types.
These findings have implications for developing patient therapies. Even though Prkci can be active in certain skin cancers, inhibiting it might lead to unintended consequences, such as tumor overgrowth. However, for patients with certain injuries or diseases, it could be therapeutic to use small molecule inhibitors to block the activity of Prkci, thus boosting stem cell production.
“We expect that our findings will be applicable in diverse contexts and make it possible to easily generate stem cells that have typically been difficult to generate,” said Mariani, principal investigator at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC.
Additional co-authors on the study include Rachel Soloff and Stephen Hedrick from UCSD. The research was supported by USC and the Robert E. and May R. Wright Foundation.
PRKCI protein kinase C, iota [ Homo sapiens (human) ]
Organism Homo sapiens Lineage – Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo
Also known asPKCI; DXS1179E; nPKC-iota Summary: This gene encodes a member of the protein kinase C (PKC) family of serine/threonine protein kinases. The PKC family comprises at least eight members, which are differentially expressed and are involved in a wide variety of cellular processes. This protein kinase is calcium-independent and phospholipid-dependent. It is not activated by phorbolesters or diacylglycerol. This kinase can be recruited to vesicle tubular clusters (VTCs) by direct interaction with the small GTPase RAB2, where this kinase phosphorylates glyceraldehyde-3-phosphate dehydrogenase (GAPD/GAPDH) and plays a role in microtubule dynamics in the early secretory pathway. This kinase is found to be necessary for BCL-ABL-mediated resistance to drug-induced apoptosis and therefore protects leukemia cells against drug-induced apoptosis. There is a single exon pseudogene mapped on chromosome X. [provided by RefSeq, Jul 2008]
A Prkci gene keeps stem cells in check
Date:
October 30, 2015
Source:
University of Southern California – Health Sciences
Summary:
When it comes to stem cells, too much of a good thing isn’t wonderful: producing too many new stem cells may lead to cancer; producing too few inhibits the repair and maintenance of the body. Medical researchers now describe a key gene in maintaining this critical balance between producing too many and too few stem cells.
Newly-discovered gene controls stem cell production
A scientific team from the University of Southern California (USC) and the University of California, San Diego have described an important gene that maintains a critical balance between producing too many and too few stem cells. Called Prkci, the gene influences whether stem cells self-renew to produce more stem cells, or differentiate into more specialized cell types, such as blood or nerves.
When it comes to stem cells, too much of a good thing isn’t necessarily a benefit: producing too many new stem cells may lead to cancer; making too few inhibits the repair and maintenance of the body.
In their experiments, the researchers grew mouse embryonic stem cells, which lacked Prkci, into embryo-like structures in the laboratory. Without Prkci, the stem cells favored self-renewal, generating large numbers of stem cells and, subsequently, an abundance of secondary structures.
Upon closer inspection, the stem cells lacking Prkci had many activated genes typical of stem cells, and some activated genes typical of neural, cardiac, and blood-forming cells. Therefore, the loss of Prkci can also encourage stem cells to differentiate into the progenitor cells that form neurons, heart muscle, and blood.
Prkci achieves these effects by activating or deactivating a well-known group of interacting genes that are part of the Notch signaling pathway. In the absence of Prkci, the Notch pathway produces a protein that signals to stem cells to make more stem cells. In the presence of Prkci, the Notch pathway remains silent, and stem cells differentiate into specific cell types.
These findings have implications for developing patient therapies. Even though Prkci can be active in certain skin cancers, inhibiting it might lead to unintended consequences, such as tumor overgrowth. However, for patients with certain injuries or diseases, it could be therapeutic to use small molecule inhibitors to block the activity of Prkci, thus boosting stem cell production.
“We expect that our findings will be applicable in diverse contexts and make it possible to easily generate stem cells that have typically been difficult to generate,” said Francesca Mariani, Ph.D., principal investigator at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC.
Their study (“Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway”) was published in a Stem Cell Reports.
Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway
In Kyoung Mah,1 Rachel Soloff,2,3 Stephen M. Hedrick,2 and Francesca V. Mariani1, *
*Correspondence: fmariani@usc.edu http://dx.doi.org/10.1016/j.stemcr.2015.09.021
The number of stem/progenitor cells available can profoundly impact tissue homeostasis and the response to injury or disease. Here, we propose that an atypical PKC, Prkci, is a key player in regulating the switch from an expansion to a differentiation/maintenance phase via regulation of Notch, thus linking the polarity pathway with the control of stem cell self-renewal. Prkci is known to influence symmetric cell division in invertebrates; however a definitive role in mammals has not yet emerged. Using a genetic approach, we find that loss of Prkci results in a marked increase in the number of various stem/progenitor cells. The mechanism used likely involves inactivation and symmetric localization of NUMB, leading to the activation of NOTCH1 and its downstream effectors. Inhibition of atypical PKCs may be useful for boosting the production of pluripotent stem cells, multipotent stem cells, or possibly even primordial germ cells by promoting the stem cell/progenitor fate.
The control of asymmetric versus symmetric cell division in stem and progenitor cells balances self-renewal and differentiation to mediate tissue homeostasis and repair and involves key proteins that control cell polarity. In the case of excess symmetric division, too many stem-cell-like daughter cells are generated that can lead to tumor initiation and growth. Conversely, excess asymmetric cell division can severely limit the number of cells available for homeostasis and repair (Go´mez-Lo´pez et al., 2014; Inaba and Yamashita, 2012). The Notch pathway has been implicated in controlling stem cell self-renewal in a number of different contexts (Hori et al., 2013). However, how cell polarity, asymmetric cell division, and the activation of determinants ultimately impinges upon the control of stem cell expansion and maintenance is not fully understood. In this study, we examine the role of an atypical protein kinase C (aPKC), PRKCi, in stem cell self-renewal and, in particular, determine whether PRKCi acts via the Notch pathway.
PKCs are serine-threonine kinases that control many basic cellular processes and are typically classified into three subgroups—conventional, novel, and the aPKCs iota and zeta, which, in contrast to the others, are not activated by diacylglyceride or calcium. The aPKC proteins are best known for being central components of an evolutionarily conserved Par3-Par6-aPKC trimeric complex that controls cell polarity in C. elegans, Drosophila, Xenopus, zebrafish, and mammalian cells (Suzuki and Ohno, 2006).
Before Notch influences stem cell self-renewal, the regulation of cell polarity, asymmetric versus symmetric cell division, and the segregation of cell fate determinants such as NUMB may first be required (Knoblich, 2008). For example, mutational analysis in Drosophila has demonstrated that the aPKC-containing trimeric complex is required for maintaining polarity and for mediating asymmetric cell division during neurogenesis via activation and segregation of NUMB (Wirtz-Peitz et al., 2008). NUMB then functions as a cell fate determinant by inhibiting Notch signaling and preventing self-renewal (Wang et al., 2006). In mammals, the PAR3-PAR6-aPKC complex also can bind and phosphorylate NUMB in epithelial cells and can regulate the unequal distribution of Numb during asymmetric cell division (Smith et al., 2007). During mammalian neurogenesis, asymmetric division is also thought to involve the PAR3-PAR6-aPKC complex, NUMB segregation, and NOTCH activation (Bultje et al., 2009).
Mice deficient in Prkcz are grossly normal, with mild defects in secondary lymphoid organs (Leitges et al., 2001). In contrast, deficiency of the Prkci isozyme results in early embryonic lethality at embryonic day (E)9.5 (Seidl et al., 2013; Soloff et al., 2004). A few studies have investigated the conditional inactivation of Prkci; however, no dramatic changes in progenitor generation were detected in hematopoietic stem cells (HSCs) or the brain (Imai et al., 2006; Sengupta et al., 2011), although one study found evidence of a role for Prkci in controlling asymmetric cell division in the skin (Niessen et al., 2013). Analysis may be complicated by functional redundancy between the iota and zeta isoforms and/or because further studies perturbing aPKCs in specific cell lineages and/or at specific developmental stages are needed. Therefore, a complete picture for the requirement of aPKCs at different stages of mammalian development has not yet emerged.
Here, we investigate the requirement of Prkci in mouse cells using an in vitro system that bypasses early embryonic lethality. Embryonic stem (ES) cells are used to make embryoid bodies (EBs) that develop like the early post-implantation embryo in terms of lineage specification and morphology and can also be maintained in culture long enough to observe advanced stages of cellular differentiation (Desbaillets et al., 2000). Using this approach, we provide genetic evidence that inactivation of Prkci signaling leads to enhanced generation of pluripotent cells and some types of multipotent stem cells, including cells with primordial germ cell (PGC) characteristics. In addition, we provide evidence that aPKCs ultimately regulate stem cell fate via the Notch pathway
Figure 1. Prkci/ EBs Contain Cells with Pluripotency Characteristics (A and A0 ) Day (d) 12 heterozygous EBs have few OCT4/E-CAD+ cells, while null EBs contain many in clusters at the EB periphery. Inset: OCT4 (nucleus)/E-CAD (cytoplasm) double-positive cells. (B and B0 ) Adjacent sections in a null EB show that OCT4+ cells are likely also SSEA1+. (C) Dissociated day-12 Prkci/ EBs contain five to six times more OCT4+ and approximately three times more SSEA1+ cells than heterozygous EBs (three independent experiments). (D and D0 ) After 2 days in ES cell culture, no colonies are visible in null SSEA1 cultures while present in null SSEA1+ cultures (red arrows). (E–E00) SSEA1+ sorted cells can be maintained for many passages, 27+. (E) Prkci+/ sorted cells make colonies with differentiated cells at the outer edges (n = 27/35). (E0 ) Null cells form colonies with distinct edges (n = 39/45). (E00) The percentage of undifferentiated colonies is shown. ***p < 0.001.
(F) Sorted null cells express stem cell and differentiation markers at similar levels to normal ES cells (versus heterozygous EBs) (three independent experiments). (G) EBs made from null SSEA1+ sorted cells express germ layer marker genes at the indicated days. Error bars indicate mean ± SEM, three independent experiments. Scale bars, 100 mm in (A, D, and E); 25 mm in (B). See also Figure S1.
Prkci/ Cultures Have More Pluripotent Cells Even under Differentiation Conditions First, we compared Prkci null EB development to that of Prkci/ embryos. Consistent with another null allele (Seidl et al., 2013), both null embryos and EBs fail to properly cavitate (Figures S1A and S1B). The failure to cavitate is unlikely to be due to the inability to form one of the three germ layers, as null EBs express germ-layer-specific genes (Figure S1E). A failure of cavitation could alternatively be caused by an accumulation of pluripotent cells. For example, EBs generated from Timeless knockdown cells do not cavitate and contain large numbers of OCT4-expressing cells (O’Reilly et al., 2011). In addition, EBs generated with Prkcz isoform knockdown cells contain OCT4+ cells under differentiation conditions (Dutta et al., 2011; Rajendran et al., 2013). Thus, we first evaluated ES colony differentiation by alkaline phosphatase (AP) staining. After 4 days without leukemia inhibitory factor (LIF), Prkci/ ES cell colonies retained crisp boundaries and strong AP staining. In contrast, Prkci+/ colonies had uneven colony boundaries with diffuse AP staining (Figures S1F–S1F00). To definitively detect pluripotent cells, day-12 EBs were assayed for OCT4 and E-CADHERIN (E-CAD) protein expression. Prkci+/ EBs had very few OCT4/E-CAD double-positive cells (Figure 1A); however, null EBs contained large clusters of OCT4/E-CAD double-positive cells, concentrated in a peripheral zone (Figure 1A0 ). By examining adjacent sections, we found that OCT4+ cells could also be positive for stage-specific embryonic antigen 1 (SSEA1) (Figures 1B and 1B0 ). Quantification by fluorescence-activated cell sorting (FACS) analysis showed that day-12 Prkci/ EBs had more OCT4+ and SSEA1+ cells than Prkci+/ EBs (Figure 1C). We did not find any difference between heterozygous and wild-type cells with respect to the number of OCT4+ or SSEA1+ cells or in their levels of expression for Oct4, Nanog, and Sox2 (Figures S1I, S1I0 and S1J). However, we did find that Oct4, Nanog, and Sox2 were highly upregulated in OCT4+ null cells (Figure S1G). Thus, together, these data indicate that Prkci/ EBs contain large numbers of pluripotent stem cells, despite being cultured under differentiation conditions.
Functional Pluripotency Tests If primary EBs have a pluripotent population with the capacity to undergo self-renewal, they can easily form secondary EBs (O’Reilly et al., 2011). Using this assay, we found that more secondary EBs could be generated from Prkci/ versus Prkci+/ EBs, especially at days 6, 10, and 16; even when plated at a low density to control for aggregation (Figure S1H). To test whether SSEA1+ cells could maintain pluripotency long term, FACS-sorted Prkci/ SSEA1+ and SSEA1 cells were plated at a low density and maintained under ES cell culture conditions. SSEA1 cells were never able to form identifiable colonies and could not be maintained in culture (Figure 1D). SSEA1+ cells, however, formed many distinct colonies after 2 days of culture, and these cells could be maintained for over 27 passages (Figures 1D0 , 1E0 , and 1E00). Prkci+/ SSEA1+ cells formed colonies that easily differentiated at the outer edge, even in the presence of LIF (Figure 1E). In contrast Prkci/ SSEA1+ cells maintained distinct round colonies (Figure 1E0 ). Next, we determined whether null SSEA1+ cells expressed pluripotency and differentiation markers similarly to normal ES cells. Indeed, we found that Oct4, Nanog, and Sox2 were upregulated in both null SSEA1+ EB cells and heterozygous ES cells. In addition, differentiated markers (Fgf5, T, Wnt3, and Afp) and tissue stem/progenitor cell markers (neural: Nestin, Sox1, and NeuroD; cardiac: Nkx2-5 and Isl1; and hematopoietic: Gata1 and Hba-x) were downregulated in both SSEA1+ cells and heterozygous ES cells (Figure 1F). SSEA1+ cells likely have a wide range of potential, since EBs generated from these cells expressed markers for all three germ layers (Figure 1G). In addition, as expected, EBs made from null SSEA1+ cells were (F) Sorted null cells morphologically abnormal, similar to the EBs made from unsorted Prkci/ ES cells (Figure S1G0 ). Thus, taken together, several assays indicate that the OCT4 and SSEA1+ populations enriched in null EBs represent pluripotent stem cells that can self-renew and have broad differentiation capacity.
ERK1/2 Signaling during EB Development Stem cell self-renewal has been shown to require the activation of the JAK/STAT3 and PI3K/AKT pathways and the inhibition of ERK1/2 and GSK3 pathways (Kunath et al., 2007; Niwa et al., 1998; Sato et al., 2004; Watanabe et al., 2006). We found that both STAT3 and phosphorylated STAT3 levels were not grossly altered and that the p-STAT3/STAT3 ratio was similar between heterozygous and null ES cells and EBs (Figures S2A and S2B). In addition we did not see any difference in AKT, pAKT, or b-CATENIN levels when comparing heterozygous to null ES cells or EBs (Figures S2A and S2C). Thus, the effects observed by the loss of Prkci are unlikely to be due to a significant alteration in the JAK/STAT3, PI3K/AKT, or GSK3 pathways.
Next, we investigated ERK1/2 expression and activation. Consistent with other studies showing ERK1/2 activation to be downstream of Prkci in some mammalian cell types (Boeckeler et al., 2010; Litherland et al., 2010), pERK1/2 was markedly inactivated in Prkci null versus heterozygous ES cells. In addition, during differentiation, null EBs displayed strong pERK1/2 inhibition early (until day 6). Later, pERK1/2 was activated strongly, as the EB began differentiating (Figures 2A and 2B). By immunofluorescence, pERK1/2 was strongly enriched in the columnar epithelium of control EBs, while overall levels were much lower in Prkci/ EBs (Figure 2C). In addition, high OCT4 expression correlated with a marked inactivation of pERK1/2 (Figure 2C). Next, we examined Prkci/ SSEA1+ cells by western blot. We found that SSEA1+ cells isolated from day-12 null EBs had pSTAT3 expression levels similar to whole EBs, while pERK1/2 levels were low (Figure 2D). Thus, these experiments indicate that the higher numbers of pluripotent cells in null EBs correlate with a strong inactivation of ERK1/2.
Figure 2. Prkci and Pluripotency Pathways (A) ERK1/2 phosphorylation (Y202/Y204) is reduced in null ES cells and early day (d)-6 null EBs compared to heterozygous EBs and strongly increased at later stages. The first lane shows ES cells activated (A) by serum treatment 1 day after serum depletion. (B) Quantification of pERK1/2 normalized to non-phosphorylated ERK1/2 (three independent experiments; mean ± SEM; **p < 0.01). (C) pERK1/2 Y202/Y204 is strongly expressed in the columnar epithelium of heterozygous EBs that have just cavitated. Null EBs have lower expression. OCT4 and pERK1/2 expression do not co-localize. Scale bar, 100 mm. (D) pERK1/2Y202/Y204 levels are lower in null SSEA1+ sorted cells than in heterozygous or in null day-12 EBs that have undergone further differentiation. pSTAT3 and STAT levels are unchanged. See also Figure S2.
Neural Stem Cell Fate Is Favored in Prkci/ EBs It is well known that ERK/MEK inhibition is not sufficient for pluripotent stem cell maintenance (Ying et al., 2008); thus, other pathways are likely involved. Therefore, we used a TaqMan Mouse Stem Cell Pluripotency Panel (#4385363) on an OpenArray platform to investigate the mechanism of Prkci action. Day 13 and day 20 Prkci/ EBs expressed high levels of pluripotency and stemness markers versus heterozygous EBs, including Oct4, Utf1, Nodal, Xist, Fgf4, Gal, Lefty1, and Lefty2. However, interestingly, EBs also expressed markers for differentiated cell types and tissue stem cells, including Sst, Syp, and Sycp3 (neural-related genes), Isl1 (cardiac progenitor marker), Hba-x, and Cd34 (hematopoietic markers). Based on this first-pass test, we sought to determine whether loss of Prkci might favor the generation of neural, cardiac, and hematopoietic cell types and/or their progenitors.
First, we found that null EBs contained many more NESTIN- and PAX6-positive cells than heterozygous EBs (Figures 3A and 3B; Figures S3A and S3B) (neural stem A and progenitor markers) (Sansom et al., 2009; Tohyama et al., 1992). In addition, quantification of PAX6 immuno- fluorescence (easier to quantify because of its nuclear localization) using a pixel count method (Fogel et al., 2012) revealed more abundant PAX6+ cells in null EBs versus heterozygous EBs. This difference was no longer evident at day 16, presumably because most of the new neural progenitors had differentiated (Figure 3D). Indeed, differentiated neuronal markers MAP2 and TUJ1 could be expressed in null cell cultures (Figures 3C and 3C0 ). Retinoic acid (RA) treatment both in EBs and ES cells promotes neurogenesis (Xu et al., 2012). We found that, even under RA induction, null cultures contained a larger population of NESTIN+ and a smaller population of TUJ1+ cells when compared to heterozygous cultures (Figures 3E and 3F). Again, null neural progenitors were capable of undergoing some differentiation, since we could find cells expressing NEUROD, NEUN, and MAP2 (Figures 3F0 –3F000). We also assessed neurogenesis in monolayer culture, using media that promotes neural stem cell generation supplemented with a low concentration of RA (Xu et al., 2012). Similar to the EB assay, we found that null ES cells generated a larger NESTIN+ and smaller TUJ1+ population compared to heterozygous ES cells (Figures S3C and S3D). Like in EBs, MAP2- and TUJ1-positive cells could still be found in the null cultures (Figure S3D0 ). Thus, using several different neural-induction assays, we found that the absence of Prkci correlates with the production of more neural progenitors and that, although these cells may favor self-renewal, they are still capable of progressing toward differentiation.
Figure 3. Neural Stem Cell Populations Are Increased in Null EBs (A–C0 ) Prkci/ EBs (B) have more NESTINpositive cells than Prkci+/ EBs (A). (C and C0 ) MAP2 and TUJ1 are expressed in null EBs, similarly to heterozygous EBs (data not shown). (D) EBs were assessed for PAX6 expression, and the images were used for quantification (Figures S3A and S3B). The pixel count ratio of PAX6+ cells in null EBs (green) is substantially higher than that found in heterozygous EBs (black) (three independent experiments; mean ± SEM; *p < 0.05). (E–F000) Day 4 after RA treatment, Prkci/ EBs have more NESTIN- than TUJ1-positive neurons (E and F). However, null cells can still terminally differentiate into NEUROD-, NEUN-, and MAP2-positive cells (F0 –F000). Scale bars, 25 mm in (A and C) and 50 mm in (E). See also Figure S3.
The Generation of Cardiomyocyte and Erythrocyte Progenitors Is Also Favored Next, we examined ISL1 expression (a cardiac stem cell marker) by immunofluorescence and found that Prkci/ EBs contained larger ISL1 clusters compared with Prkci+/ EBs; this was confirmed using an image quantification assay (Figures 4A, 4A0 , and 4C). Differentiated cardiac cells and ventral spinal neurons can also express ISL1 (Ericson et al., 1992); therefore, we also examined Nkx2-5 expression, a better stem cell marker and regulator of cardiac progenitor determination (Brown et al., 2004), by RT-PCR and immunofluorescence. In null EBs, Nkx2-5 was upregulated (Figure 4D). In addition, in response to RA, which can promote cardiac fates in vitro (Niebruegge et al., 2008), cells expressing NKX2-5 were more prevalent in null versus heterozygous EBs (Figures 4B and 4B0 ).The abundant cardiac progenitors found in null EBs were still capable of undergoing differentiation (Figures 4E–4F0 ). Indeed, more cells exhibited the striated pattern characteristic of a-ACTININ in null versus heterozygous EBs with RA induction (Figures 4F and 4F0 ). In addition, many more Prkci/ EBs were beating after days 6 and 12 of culture (Figure 4G).
Figure 4. Cardiomyocyte and Erythrocyte Progenitors Are Increased in Prkci/ EBs (A–F0 ) In (A, A0 , E, and E0 ), Prkci/ EBs cultured without LIF have more ISL1 (cardiac progenitor marker) and a-ACTININ-positive cells compared to heterozygous EBs. (C) At day (d) 9, the pixel count ratio for ISL1 expression indicates that null EBs (green) have larger ISL1 populations than heterozygous EBs (black) (three independent experiments, n = 20 heterozygous EBs, 21 null EBs total; mean ± SEM; *p < 0.05). In (B, B0 , D, F, and F0 ), RA treatment induces more NKX2-5 (both nuclear and cytoplasmic) and a-ACTININ expression in null EBs. Arrows point to fibers in (F0 ). (G) Null EBs (green) generate more beating EBs with RA treatment compared to heterozygous EBs (black) (four independent experiments; mean ± SEM; *p < 0.05, ***p < 0.001). (H) Dissociated null EBs of different stages (green) generate more erythrocytes in a colony-forming assay (CFU-E) (four independent experiments; mean ± SEM; **p < 0.01). (I) Examples of red colonies. (J) Gene expression for primitive HSC markers is upregulated in null EBs (relative to heterozygous EBs) (three independent experiments; mean ± SEM). Scale bars, 50 mm in (A, B, and E); 100 mm in (F), and 25 mm in (I). See also Figure S4.
Hba-x expression is restricted to yolk sac blood islands and primitive erythrocyte populations (Lux et al., 2008; Trimborn et al., 1999). Cd34 is also a primitive HSC marker (Sutherland et al., 1992). Next, we determined whether the elevated expression of these markers observed with OpenArray might represent higher numbers of primitive hematopoietic progenitors. Using a colony-forming assay (Baum et al., 1992), we found that red colonies (indicative of erythrocyte differentiation; examples in Figure 4I) were produced significantly earlier and more readily from cells isolated from null versus heterozygous EBs (Figure 4H). By quantitative real-time PCR, upregulation of Hba-x and Cd34 genes confirmed the OpenArray results (Figure 4J). In addition, we found Gata1, an erythropoiesis-specific factor, and Epor, an erythropoietin receptor that mediates erythroid cell proliferation and differentiation (Chiba et al., 1991), to be highly upregulated in null versus heterozygous EBs (Figure 4J). These data suggest that the loss of Prkci promotes the generation of primitive erythroid progenitors that can differentiate into erythrocytes.
To determine whether the aforementioned tissue stem cells identified were represented in the OCT4+ population that we described earlier, we examined the expression of PAX6, ISL1, and OCT4 in adjacent EB sections. We found that cells expressing OCT4 appeared to represent a distinct population from those expressing PAX6 and ISL1 (although some cells were PAX6 and ISL1 double-positive) (Figures S4A–S4C).
Prkci/ Cells Are More Likely to Inherit NUMB/aNOTCH1 Symmetrically The enhanced production of both pluripotent and tissue stem cells suggests that the mechanism underlying the action of Prkci in these different contexts is fundamentally similar. Because the Notch pathway controls stem cell self-renewal in many contexts (Hori et al., 2013), and because previous studies implicated a connection between PRKCi function and the Notch pathway (Bultje et al., 2009; Smith et al., 2007), we examined the localization and activation of a key player in the Notch pathway, NUMB, (Inaba and Yamashita, 2012). Differences in NUMB expression were first evident in whole EBs, where polarized expression was evident in the ectodermal and endodermal epithelia of heterozygous EBs, while Prkci/ EBs exhibited a more even distribution (Figures 5A–5B0 ). To more definitively determine the inheritance of NUMB during cell division, doublets undergoing telophase or cytokinesis were scored for symmetric (evenly distributed in both cells) or asymmetric (unequally distributed) NUMB localization (examples: Figures 5C and 5C0 ). In dissociated day-10 EBs, Prkci+/ doublets displayed somewhat less symmetric versus asymmetric inheritance, while Prkci/ doublets exhibited nearly four times more symmetric versus asymmetric inheritance (Figure 5D). Although individual cells from null EBs that were OCT4+ or PAX6+ more likely to exhibit non-polarized NUMB distribution (Figures S5A and S5B), we decided to use an assay that allowed for FACS purifi- cation, followed by the more stringent doublet assay. Therefore, we chose CD24 (heat-stable antigen; BA-1), a cell-surface marker that is highly expressed in pre-differentiated neurons and neuroblasts (Pruszak et al., 2009), and tested this marker as a method to enrich for cells destined to differentiate into neurons (see Supplemental Experimental Procedures). To assess NUMB localization, FACSsorted CD24 cells isolated from the RA-treated EBs were then put in culture for 24 hr, and doublets were scored. Both Prkci/ CD24high and CD24low doublets exhibited more symmetric versus asymmetric NUMB localization when compared to Prkci+/ doublets (Figure 5E) (>23 more was observed for CD24low doublets; 1.5 ± 0.25 [null] versus 0.67 ± 0.2 [heterozygous]). Thus, in summary, loss of Prkci favors the generation of cells with symmetric NUMB distribution, even during EB differentiation. In addition, in situations where neurogenesis is stimulated (RA treatment), loss of Prkci favors symmetric NUMB distribution in both the CD24high/low subpopulations.
Because NUMB can be directly phosphorylated by aPKCs (both PRKCi and PRKCz) (Smith et al., 2007; Zhou et al., 2011), loss of Prkci might be expected to lead to decreased NUMB phosphorylation. Three NUMB phosphorylation sites—Ser7, Ser276, and Ser295—could be aPKC mediated (Smith et al., 2007). By immunofluorescence, we found that one of the most well-characterized sites (Ser276), was strongly inactivated in null versus heterozygous EBs, especially in the core (Figures 5F and 5G). Western analysis also confirmed that the levels of pNUMB (Ser276) were decreased in null versus heterozygous EBs (Figure S5F). Thus, genetic inactivation of Prkci leads to a marked decrease in the phosphorylation status of NUMB. Notch pathway inhibition by NUMB has been observed in flies and mammals (Berdnik et al., 2002; French et al., 2002). Therefore, we investigated whether reduced Numb activity in Prkci/ EBs might lead to enhanced NOTCH1 activity and the upregulation of the downstream transcriptional readouts (Meier-Stiegen et al., 2010). An overall increase in NOTCH1 activation was supported by western blot analysis showing that the level of activated NOTCH1 (aNOTCH1) was strongly increased in day 6 and day 10 null versus heterozygous EBs (Figure S5G). This was supported by immunofluorescence in EBs, where widespread strong expression of aNOTCH1 was seen in most null cells (Figures 5I and 5I0 ), while in heterozygous EBs, this pattern was observed only in the OCT4+ cells (Figures 5H and 5H0 ).
Figure 5. Prkci/ Cells Preferentially Inherit Symmetric Localization of NUMB and aNOTCH1 and Notch Signaling Is Required for Stem Cell Self-Renewal in Null Cells (A–B0 ) In (A and B), day (d)-7 heterozygous EBs have polarized NUMB localization within epithelia and strong expression in the endoderm, while null EBs have a more even distribution. (A0 and B0 ) Enlarged views. (C and C0 ) Asymmetric and symmetric NUMB expression examples. (D) Doublets from day-10 null EBs have more symmetric inheritance when compared to day-10 heterozygous doublets (three independent experiments; mean ± SEM; **p < 0.01). A red line indicates a ratio of 1 (equal percent symmetric and asymmetric). (E) CD24 high null doublets exhibited more symmetric NUMB inheritance than CD24 high heterozygous doublets (three independent experiments; mean ± SEM; *p < 0.05). A red line indicates where the ratio is 1. (F and G) Decreased pNUMB (Ser276) is evident in the core of null versus heterozygous EBs (n = 10 of each genotype). (H–I0 ) In (H and I), aNOTCH1 is strongly expressed in heterozygous EBs, including both OCT4+ and OCT4 cells, while strong aNOTCH1 expression is predominant in OCT4+ cells of null EBs (n = 10 of each genotype)). (H0 and I0 ) Enlarged views of boxed regions. OCT4+ cells are demarcated with dotted lines. (J and J0 ) OCT4+ cells express HES5 strongly in the nucleus (three independent experiments). (K) Null doublets from dissociated EBs have more symmetric aNOTCH1 inheritance compared to heterozygous doublets (three independent experiments; mean ± SEM; **p < 0.01). A red line indicates where the ratio is 1. (L) CD24high Prkci/ doublets exhibit more symmetric aNOTCH1 than CD24high heterozygous doublets (three independent experiments; mean ± SEM; *p < 0.05). A red line indicates where the ratio is 1. (M and M0 ) Examples of asymmetric and symmetric aNOTCH1 localization. (N and O) Day-3 DMSO-treated null ES colonies show strong AP staining all the way to the colony edge in (N). Treatment with 3 mM DAPT led to more differentiation in (O). (P–R) OCT4 is strongly expressed in day-4 DMSO-treated null ES cultures (P). With DAPT (Q,R), OCT4 expression is decreased. (S) Working model: In daughter cells that undergo differentiation, PRKCi can associate with PAR3 and PAR6. NUMB is recruited and directly phosphorylated. The activation of NUMB then leads to an inhibition in NOTCH1 activation and stimulation of a differentiation/maintenance program. In the absence of Prkci, the PAR3/PAR6 complex cannot assemble (although it may do so minimally with Prkcz). NUMB asymmetric localization and phosphorylation is reduced. Low levels of pNUMB are not sufficient to block NOTCH1 activation, and activated NOTCH1 preserves the stem cell self-renewal program. We suggest that PRKCi functions to drive differentiation by pushing the switch from an expansion phase that is symmetric to a differentiation and/or maintenance phase that is predominantly asymmetric. In situations of low or absent PRKCi, we propose that the expansion phase is prolonged. Scale bars, 50 mm in (A, B, F, G, H, I, J, J0 , P–R); 200 mm in (A0 and B0 ); 25 mm in (C, C0 , M, and M0 ); and 100 mm in (H0 , I0 , N, and O). See also Figure S5.
To examine the localization of aNOTCH1 and to better quantify the results seen in Figures 5H and 5I, doublets from dissociated EBs were scored. As seen with NUMB localization, null doublets were more likely to have symmetric localization of aNOTCH1 in comparison to heterozygous doublets (Figure 5K; examples in Figures 5M and 5M0 ). In addition, both CD24high and CD24low doublets from RA-treated null EBs were more likely to exhibit symmetric aNOTCH1 distribution versus doublets from RA-treated heterozygous EBs (Figure 5L; 3.46 ± 0.8 [null] versus 0.59 ± 0.06 [heterozygous] in CD24low doublets). In addition, by RT-PCR, the expression of Notch downstream genes Hes1, Hes5, Hey1, and Hey2 was increased in null versus heterozygous EBs (Figure S5I). Furthermore, HES5 by immunofluorescence was broadly expressed at similar levels in both null and heterozygous cells (Figures 5J and 5J0 ; Figures S5H and S5H0 ) but more strongly expressed in null OCT4+ cells (Figures 5J and 5J0 ). Thus, loss of Prkci is associated with NOTCH1 activation, aNOTCH1 symmetric localization, and the upregulation of Hes/Hey downstream genes in several assays.
To determine whether Notch pathway activation is required in the absence of Prkci, we examined AP activity and OCT4 expression while blocking the Notch pathway using DAPT to inhibit g-secretase (Sastre et al., 2001). DMSO-treated null ES cells stayed undifferentiated (sharp-edged colonies, strong AP staining); however, treatment of null ES cells with 3 mM DAPT led to more differentiation (AP-negative cells with cellular extensions) (Figures 5N, 5O, and S5J). In addition, OCT4 is strongly expressed in day-4 control ES cell cultures; however, in the presence of DAPT, OCT4 expression is much decreased both in monolayer culture (Figures 5P–5R) and in null EBs (48% lower OCT4+ signal versus DMSO controls, pixel counting on EB sections; data not shown). These results support the idea that activated Notch signaling is required in the absence of Prkci to see enhanced pluripotency.
Taken together, the combined effects of decreased NUMB activation, favored symmetric distribution of NUMB and aNOTCH1 and increased NOTCH1 activity support a model whereby loss of Prkci leads to sustained generation of pluripotent and some tissue stem cell populations (Figure 5S; and see Discussion).
Additional Loss of PRKCz Activity Boosts the Number of OCT4-, SSEA1-, and STELLA-Positive Cells The generation and maintenance of pluripotent stem cells from new sources or tissue stem cells for basic or translational research can be challenging, and there is need for new in vitro strategies. A PKC inhibitor (Go¨6983) that inhibits PKCa, -b, -g, -d, and -z has been used to help maintain mouse and rat ES cells in the absence of LIF (Dutta et al., 2011; Rajendran et al., 2013). Thus, we hypothesized that treating null cells with Go¨6983 might lead to better stem cell expansion compared to loss of just Prkci. In our hands, we found that, under differentiation conditions (no LIF), heterozygous ES cells treated with the inhibitor for 4 days still underwent differentiation (Figure 6A), while treated null ES cells largely stayed undifferentiated (Figure 6A0 ; Figure S6A). Drug treatment of heterozygous EBs boosted the generation of OCT4-expressing cells (Figure 6B), while treatment of null EBs resulted in an even larger OCT4+ population (Figure 6B0 ). NUMB localization was also moderately affected (Figure S6B). By cell sorting, we found that drug treatment significantly increased the percentage of OCT4+ cells in both Prkci+/ and Prkci/ EBs (Figures 6C and 6C0 ; Figures S6C and S6C0 ). Interestingly, Go¨6983 treatment also boosted the generation of SSEA1+ cells in both null and heterozygous EBs (Figures 6D and 6D0 ; Figures S6D and S6D0 ).
SSEA1 is expressed in BLIMP1-positive PGCs derived from mouse epiblast stem cells (Hayashi and Surani, 2009). Also, PGC-like cells can be derived from isolated SSEA1+/OCT4+ EB cells (Geijsen et al., 2004). Therefore, we speculated that the increase in SSEA1 and OCT4 due to Go¨6983 treatment could represent an increase in the generation of PGC-like cells instead of undifferentiated ES cells. Therefore, we examined the expression of STELLA (a PGC marker). As expected, heterozygous EBs contain small clusters of STELLA+ cells similar to EBs made of wild-type cells (Figure 6E) (Payer et al., 2006). The addition of Go¨6983 to Prkci+/ EBs induced a modest increase in the number of STELLA+ cells present in the clusters (Figure 6F). Without drug treatment, null EBs contained more clusters, and the clusters contained more STELLA+ cells when compared to heterozygous EBs (Figures 6E and 6G). Interestingly, when Prkci/ EBs were treated with Go¨6983, the generation of STELLA+ cells was strongly enhanced (Figure 6G versus Figure 6H). Because undifferentiated ES cells can still express STELLA (Payer et al., 2006), we co-stained Prkc EBs for VASA (a more differentiated PGC marker). We found many cells that were double positive (a little less than half) (Figure 6K) but also cells that expressed VASA only and STELLA only (23 more than VASA only) (Figures 6I–6K, red/green arrows). Therefore, the combined effect of loss of Prkci and PKC inhibition via Go¨6983 treatment leads to the production of STELLA and VASA+ PGC-like cells.
Figure 6. Additional Inhibition of PRKCz Results in an Even Higher Percentage of OCT4-, SSEA1-, and STELLA-Positive Cells (A and A0 ) After day 4 without LIF, heterozygous ES cells undergo differentiation in the presence of Go¨6983, while null ES cells stay as distinct colonies in (A0 ). (B and B0 ) Go¨6983 stimulates an increase in OCT4+ populations in heterozygous EBs and an even larger OCT4+ population in null EBs in (B0 , insets: green and red channels separately). (C–D0 ) An even higher percentage of cells are OCT4+ (C and C0 ) and SSEA1+ (D and D0 ) with Go¨6983 treatment (day 12, three independent experiments). (E and F) More STELLA+ clusters containing a larger number of cells are present in drugtreated heterozygous EBs. (G and H) Null EBs also have more STELLA+ clusters and cells. Drug-treated null EBs exhibit a dramatic increase in the number of STELLA+ cells. (I–K) Some cells are double positive for STELLA and VASA in drug-treated null EBs (yellow arrows). There are also VASAonly (green arrows) and STELLA-only cells (red arrows) (three independent experiments). (L–P) Treatment with ZIP results in an increase in OCT4+ and STELLA+ cells. ZIP treatment also results in more cells that are VASA+ (three independent experiments); n = 11 for Prkci+/, and n = 13 for Prkci+/ + ZIP; n = 14 for Prkci/, and n = 20 for Prkci/ + ZIP; eight EBs assayed for both STELLA and VASA expression). Scale bars, 100 mm in (A and A0 ); 50 mm in (B and B0 ); and 25 mm in (E, I, and L). See also Figure S6. 10 S.
Next, we examined whether the more specific aPKC inhibitor, ZIP, a myristolated aPKC pseudosubstrate with competitive binding to p62, had similar effects (Price and Ghosh, 2013; Tsai et al., 2015; Yao et al., 2013). We found that both heterozygous and null EBs treated with ZIP contained more OCT4+ cells compared to un-treated EBs (Figures 6L–6O). In addition, like Go¨6983, ZIP treatment resulted in a modest increase in the percentage of SSEA1+ cells found in heterozygous EBs and a strong increase in the percentage of SSEA1+ cells in null EBs (Figures S6E– S6F0 ). Furthermore, like Go¨6983, both STELLA+ and VASA+ populations were increased with ZIP treatment (Figure 6P). Thus, both pluripotent and PGC-like cells can be abundantly generated with Go¨6983 or ZIP treatment, suggesting that strategies that inhibit both PRKCi and/or PRKCz may be useful to maintain stem cell self-renewal and/or generate abundant PGC-like cells.
DISCUSSION In this report, we suggest that Prkci controls the balance between stem cell expansion and differentiation/maintenance by regulating the activation of NUMB, NOTCH1, and Hes /Hey downstream effector genes. In the absence of Prkci, the pluripotent cell fate is favored, even without LIF, yet cells still retain a broad capacity to differentiate. In addition, loss of Prkci results in enhanced generation of tissue progenitors such as neural stem cells and cardiomyocyte and erythrocyte progenitors. In contrast to recent findings on Prkcz (Dutta et al., 2011), loss of Prkci does not appear to influence STAT3, AKT, or GSK3 signaling but results in decreased ERK1/2 activation. We hypothesize that, in the absence of Prkci, although ERK1/2 inhibition may be involved, it is the decreased NUMB phosphorylation and increased NOTCH1 activation that promotes stem and progenitor cell fate. Thus, we conclude that PRKCi, a protein known to be required for cell polarity, also plays an essential role in controlling stem cell fate and generation via regulating NOTCH1 activation.
Notch Activation Drives the Decision to Self-Renew versus Differentiate Notch plays an important role in balancing stem cell selfrenewal and differentiation in a variety of stem cell types and may be one of the key downstream effectors of Prkci signaling. Sustained Notch1 activity in embryonic neural progenitors has been shown to maintain their undifferentiated state (Jadhav et al., 2006). Similarly, sustained constitutive activation of NOTCH1 stimulates the proliferation of immature cardiomyocytes in the rat myocardium (Collesi et al., 2008). In HSCs, overexpression of constitutively active NOTCH1 in hematopoietic progenitors and stem cells supports both primitive and definitive HSC selfrenewal (Stier et al., 2002). Together, these studies suggest that activation and/or sustained Notch signaling can lead to an increase in certain tissue stem cell populations. Thus, a working model for how tissue stem cell populations are favored in the absence of Prkci involves a sequence of events that ultimately leads to Notch activation. Recent studies have shown that aPKCs can be found in a complex with NUMB in both Drosophila and mammalian cells (Smith et al., 2007; Zhou et al., 2011); hence, in our working model (Figure 5S), we propose that the localization and phosphorylation of NUMB is highly dependent on the activity of PRKCi. When Prkci is downregulated or absent (as shown here), cell polarity is not promoted, leading to diffuse distribution and decreased phosphorylation of NUMB. Without active NUMB, NOTCH1 activation is enhanced, Hes/Hey genes are upregulated, and stem/progenitor fate generation is favored. To initiate differentiation, polarization could be stochastically determined but could also be dependent on external cues such as the presentation of certain ligands or extracellular matrix (ECM) proteins (Habib et al., 2013). When PRKCi is active and the cell becomes polarized, a trimeric complex is formed with PRKCi, PAR3, and PAR6. Numb is then recruited and phosphorylated, leading to Notch inactivation, the repression of downstream Hes/Hey genes, and differentiation is favored (see Figure 5S). Support for this working model comes from studies in Drosophila showing that the aPKC complex is essential for Numb activation and asymmetric localization (Knoblich, 2008; Smith et al., 2007; Wang et al., 2006). Additional studies on mouse neural progenitors show that regulating Numb localization and Notch activation is critical for maintaining the proper number of stem/progenitor cells in balance with differentiation (Bultje et al., 2009). Thus, an important function for PRKCi may be to regulate the switch between symmetric expansion of stem/progenitor cells to an asymmetric differentiation/maintenance phase. In situations of low or absent PRKCi, we propose that the expansion phase is favored. Thus, temporarily blocking either, or both, of the aPKC isozymes may be a powerful approach for expanding specific stem/progenitor populations for use in basic research or for therapeutic applications.
These studies, together with data presented here, provide genetic evidence that evolutionarily conserved polarity pathways may play a central role in NOTCH1 activation. and stem cell self-renewal in mammals. Further genetic studies using Cre transgenes that are specific for progenitors in the neural plate, primitive erythrocytes, cardiomyocytes, and other progenitors to ablate aPKC function will be needed to determine how generally this mechanism is used in diverse tissues.
Although we do not see changes in the activation status of the STAT3, AKT, or GSK3 pathway, loss of Prkci results in an inhibition of ERK1/2 (Figures 2A and 2B). This result is consistent with the findings that ERK1/2 inhibition is both correlated with and directly increases ES cell selfrenewal (Burdon et al., 1999). Modulation of ERK1/2 activity by Prkci has been observed in cancer cells and chondrocytes (Litherland et al., 2010; Murray et al., 2011). Although it is not clear whether a direct interaction exists between Prkci and ERK1/2, Prkcz directly interacts with ERK1/2 in the mouse liver and in hypoxia-exposed cells (Das et al., 2008; Peng et al., 2008). The Prkcz isozyme is still expressed in Prkci null cells but evidently cannot suf- ficiently compensate and activate the pathway normally. Furthermore, knocking down Prkcz function in ES cells does not result in ERK1/2 inhibition, suggesting that this isozyme does not impact ERK1/2 signaling in ES cells (Dutta et al., 2011). Therefore, although PRKCi may interact with ERK1/2 and be directly required for its activation, ERK1/2 inhibition could also be a readout for cells that are more stem-like. Further studies will be needed to address this question.
Utility of Inhibiting aPKC Function Loss of Prkci resulted in EBs that contained slightly more STELLA+ cells than EBs made from +/ cells. Furthermore, inhibition of both aPKC isozymes by treating Prkci null cells with the PKC inhibitor Go¨6983 or the more specific inhibitor, ZIP, strongly promoted the generation of large clusters of STELLA+ and VASA+ cells, suggesting that inhibition of both isozymes is important for PGC progenitor expansion (Figure 6). It is unclear what the mechanism for this might be; however, one possibility is that blocking both aPKCs is necessary to promote NOTCH1 activation in PGCs or in PGC progenitor cells that may ordinarily have strong inhibitions to expansion (Feng et al., 2014). Regardless of mechanism, the ability to generate PGC-like cells in culture is notoriously challenging, and our results provide a method for future studies on PGC specification and differentiation.
Expansion of stem/progenitor pools may not be desirable in the context of cancer. Prkci has been characterized as a human oncogene, a useful prognostic cancer marker, and a therapeutic target for cancer treatment. Overexpression of Prkci is found in epithelial cancers (Fields and Regala, 2007), and Prkci inhibitors are being evaluated as candidate cancer therapies (Atwood et al., 2013; Mansfield et al., 2013). However, because our results show that Prkci inhibition leads to enhanced stem cell production in vitro, Prkci inhibitor treatment as a cancer therapy might lead to unintended consequences (tumor overgrowth), depending on the context and treatment regimen. Thus, extending our findings to human stem and cancer stem cells is needed. In summary, here, we demonstrate that loss of Prkci leads to the generation of abundant pluripotent cells, even under differentiation conditions. In addition, we show that tissue stem cells such as neural stem cells, primitive erythrocytes, and cardiomyocyte progenitors can also be abundantly produced in the absence of Prkci. These increases in stem cell production correlate with decreased NUMB activation and symmetric NUMB localization and require Notch signaling. Further inhibition of Prkcz may have an additive effect and can enhance the production of PGC-like cells. Thus, Prkci (along with Prkcz) may play key roles in stem cell self-renewal and differentiation by regulating the Notch pathway. Furthermore, inhibition of Prkci and or Prkcz activity with specific small-molecule inhibitors might be a powerful method to boost stem cell production in the context of injury or disease.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, six figures, and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr. 2015.09.021.
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USC offers a summer of stem cells for local high school students
The teens boost their scientific IQ by conducting research in USC labs
The goal of these unique programs is to educate bright young minds at the stage where they’re still formulating ideas and still open and receptive to new discoveries.
Andrew McMahon
Twenty-three local high school students spent their summer vacations in a very unusual place: the Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC.
The students celebrated their graduations this month from the USC Early Investigator High School (EiHS) and the USC CIRM Science, Technology and Research (STAR) programs. These are the only programs that offer comprehensive training in stem cell research to high school students.
“The goal of these unique programs is to educate bright young minds at the stage where they’re still formulating ideas and still open and receptive to new discoveries, and introduce them to the wonder and inspirational power of stem cell biology,” said Andrew McMahon, director of USC’s stem cell research center and the Department of Stem Cell Biology and Regenerative Medicine, and head of the university-wide USC Stem Cell initiative uniting more than 100 researchers from all disciplines.
Stem cells, ethics and public policy
Over the course of the summer, the high school students participated in either a 10-day training course or eight-week research internship, working with human stem cells in USC’s world-class laboratories. Under the mentorship of USC faculty and graduate students, the students learned about the latest advances in regenerative medicine and explored stem cells, ethics and public policy.
Roberta Diaz Brinton, director of the CIRM STAR program, paid tribute to the accomplishments of the students.
“We’re very impressed by the caliber of science and more impressed by the caliber of young minds. These young scientists are generating the new knowledge from which stem cell biology and stem cell therapies will progress in the future,” said Brinton, professor at the USC School of Pharmacy, the USC Viterbi School of Engineering and the Keck School of Medicine of USC, and an executive committee member of USC Stem Cell.
True teamwork
Victoria Fox, director of the EiHS program, extended her thanks to everyone who contributed to the experience.
“The EiHS program was made possible by a team of very incredible people that starts with my laboratory staff and includes donors, the students, the administrators of the stem cell research center and the mentors who take the students in their laboratories,” she said. “I’m very grateful to all of these people.”
This year’s participants were selected from Harvard-Westlake School, Lifeline Education Charter School, Chadwick School and Bravo Medical Magnet High School, and many received scholarships.
“The program has motivated our students to be college-ready by giving them the opportunity to work in a university setting,” said Obed Nartey, principal of Lifeline Education Charter School. “Many of these students are the first generation to graduate from high school. For these students, college was seen as being out of reach until they met and worked with Dr. Fox and her team.”
On graduation day, the students shared their transformative summer experiences with their mentors, friends, parents and teachers by presenting scientific posters and by contributing articles to the program’s new EiHS Journal, which will publish its first issue in October.
“Being able to contribute to a scientific project that can play an important role in someone’s life is an amazing opportunity, and I would not trade it for the world,” said Marialuisa Flores, a student from Lifeline Education Charter School. “It was a very enjoyable learning experience, which has made a great impact on my life and future career.”
“Being able to contribute to a scientific project that can play an important role in someone’s life is an amazing opportunity, and I would not trade it for the world,” said Marialuisa Flores, a student from Lifeline Education Charter School. “It was a very enjoyable learning experience, which has made a great impact on my life and future career.”
The two-day, overnight retreat featured a plenary lecture by Clive Svendsen, director of the Regenerative Medicine Institute at Cedars-Sinai Medical Center, about the contribution of induced pluripotent stem (iPS) cells to regenerative medicine, particularly to studying and developing treatments for neurological disorders.
The retreat also included presentations by winners of the first Regenerative Medicine Initiative (RMI) Awards, which provide up to two years of seed funding for multi-investigator research collaborations that harness the full potential of USC-affiliated faculty members. The three winning teams are using various stem/progenitor cells that might lead to future therapies for certain forms of deafness, bone defects and pediatric leukemia.
Many other principal investigators, postdoctoral and graduate students shared innovative research advancing several key areas of regenerative medicine.
Rong Lu, who will leave Stanford University to join USC’s stem cell research center as a principal investigator in January, talked about her new cellular “tracking system” for hematopoietic, or blood-forming stem cells. The system allows for the more effective study of blood and other types of cancers.
Min Yu, who will leave Massachusetts General Hospital at Harvard Medical School to accept a joint appointment as a principal investigator at USC’s stem cell research center and the USC Norris Comprehensive Cancer Center in January, discussed how to filter out circulating cancer stem cells from billions of other blood cells to understand and stop cancer’s spread.
USC research associate Hu Zhao and research assistants Yichen Li and Yingxiao Shi gave presentations.
Postdoctoral students who presented research included Mohamed Hammad, Lori O’Brien, Sandeep Paul and Saaket Varma.
PhD student presenters included Wen-Hsuan Chang, Guanyi Huang, Sapna Jain, Erin Moran, Marie Rippen and Yuki Yamaguchi.
The retreat also showcased the USC stem cell research center’s core facilities for stem cell sorting, derivation, culture, iPS programming, imaging and therapeutic screening.
During the cocktail hour, guests exchanged new ideas while voting on their favorite posters, which introduced research opportunities related to the Development, Stem Cells, and Regenerative Medicine PhD program.
Retreat sponsors included the California Institute for Regenerative Medicine Amgen, Sanofi, Zeiss, Leica Microsystems, Fluidigm, Lonza and Novogenix Laboratories LLC.
“This year’s retreat was a great success,” said Andrew McMahon, who spearheads the USC Stem Cell initiative and directs the Broad Center. “It helped solidify USC Stem Cell as an interactive scientific community and build relationships with our colleagues at the university and beyond.”
$1.5M Goes to Stem Cell Research
$6.4M for Stem Cell Labs to USC, CHLA
$25 million Broad Foundation gift creates stem cell institute at USC
McMahon discusses central role of stem cell biology in medicine of the future
McMahon installed as chair of stem cell biology
Army Research Laboratory selects USC institute as base for breakthroughs in science and technology
Brainpower applied to understanding of neural stem cells
How do humans and other mammals get so brainy? USC researcher Wange Lu and his colleagues shed new light on this question in a paper published in the journal Cell Reports on Oct. 24.
The researchers donned their thinking caps to explain how neural stem and progenitor cells differentiate into neurons and related cells called glia. Neurons transmit information through electrical and chemical signals; glia surround, support and protect neurons in the brain and throughout the nervous system. Glia do everything from holding neurons in place to supplying them with nutrients and oxygen to protect them from pathogens.
By studying the embryo neural stem cells of mice in a petri dish, Lu and his colleagues discovered that a protein called SMEK1 promotes the differentiation of neural stem and progenitor cells. At the same time, SMEK1 keeps these cells in check by suppressing their uncontrolled proliferation.
The researchers also determined that SMEK1 doesn’t act alone: It works in concert with Protein Phosphatase 4 to suppress the activity of PAR3, a third protein that discourages neurogenesis — the birth of new neurons. With PAR3 out of the picture, neural stem cells and progenitors are free to differentiate into new neurons and glia.
“These studies reveal the mechanisms of how the brain keeps the balance of stem cells and neurons when the brain is formed,” said Wange Lu, associate professor of biochemistry and molecular biology at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC. “If this process goes wrong, it leads to cancer or mental retardation or other neurological diseases.”
Neural stem and progenitor cells offer tremendous promise as a future treatment for neurodegenerative disorders, and understanding their differentiation is the first step toward harnessing the cells’ therapeutic potential. This could offer new hope for patients with Alzheimer’s, Parkinson’s and many other currently incurable diseases.
Co-authors from the Broad Center included Vicky Yamamoto, Si Ho Choi and Zhong Wei. Co-authors Hee-Ryang Kim and Choun-Ki Joo work at the Catholic University of Korea in Seoul, and first author Jungmook Lyu is affiliated with both institutions.
Funding for the study came from the National Institutes of Health (grant number 5R01NS067213).
Protein phosphatase 4 and Smek complex negatively regulate Par3 and promote neuronal differentiation of neural stem/progenitor cells.
Neural progenitor cells (NPCs) are multipotent cells that can self-renew and differentiate into neurons and glial cells. However, mechanisms that control their fate decisions are poorly understood. Here, we show that Smek1, a regulatory subunit of the serine/threonine protein phosphatase PP4, promotes neuronal differentiation and suppresses the proliferative capacity of NPCs. We identify the cell polarity protein Par3, a negative regulator of neuronal differentiation, as a Smek1 substrate and demonstrate that Smek1 suppresses its activity. We also show that Smek1, which is predominantly nuclear in NPCs, is excluded from the nucleus during mitosis, allowing it to interact with cortical/cytoplasmic Par3 and mediate its dephosphorylation by the catalytic subunit PP4c. These results identify the PP4/Smek1 complex as a key regulator of neurogenesis.
Neural stem and progenitor cells located in the ventricular zone (VZ) of the embryonic neocortex are mitotically active, self-renewing cells with the potential to produce differentiated cell types (Temple, 2001). During cortical development, postmitotic neurons generated from NPCs migrate radially out of the VZ and form the cortical plate (CP) in an “inside-out pattern,” eventually establishing a six-layered cortex (Kriegstein et al., 2006). The timing of neuronal differentiation determines the size of the progenitor pool, the final number of neurons, and cortical thickness. However, the molecular mechanisms that control the switch from proliferation to neuronal differentiation of NPCs remain incompletely understood.
Studies of Drosophila neuroblasts show that the serine/threonine protein phosphatase 2A (PP2A) inhibits self-renewal and promotes neuronal differentiation by regulating the phosphorylation status of cell fate determinants, including Numb (Wang et al., 2009). Bazooka, a key component of the Par protein complex, is a well-characterized PP2A substrate in Drosophila neuroblasts (Krahn et al., 2009; Ogawa et al., 2009). PP2A antagonizes phosphorylation of Bazooka by Par1 kinase to control its subcellular localization. In mammals, a protein called Partitioning-defective 3 (Par3), the ortholog of Bazooka, accumulates at the tip of a growing axon in neurons and controls axon specification (Shi et al., 2003). Recently, it has been shown that Par3, which is enriched in the apical domain of NPCs of the VZ (Imai et al., 2006), critically regulates proliferation versus differentiation during cortical development (Bultje et al., 2009; Costa et al., 2008).
PP4, which belongs to the PP2A family, is a protein complex comprised of a catalytic subunit PP4c plus regulatory subunits (Gingras et al., 2005). Smek (also termed PP4R3) has been identified as a PP4 regulatory subunit and implicated in activities as diverse as regulation of MEK (Mendoza et al., 2005), insulin/IGF-1 signaling (Wolff et al., 2006), H2AX phosphorylation (Chowdhury et al., 2008), and histone H3 and H4 acetylation (Lyu et al., 2011). A recent study reported that Falafel (Flfl), the Drosophila homolog of Smek, mediates localization of the adaptor protein Miranda and the cell fate determinant Prospero in neuroblasts (Sousa-Nunes et al., 2009). However, the direct substrate of Smek remains unclear. Here we identify Par3 as a direct substrate of the PP4/Smek1 complex in NPCs and report a novel role for Smek1 in regulating neuronal differentiation.
Smek1 is required for neuronal differentiation of NPCs
During mouse cortical development, Smek1 is expressed in a distinct temporal and spatial pattern. At E11.5, we observed that Smek1 protein is expressed in most NPCs at the apical side of the forebrain VZ (Figure 1Aand S1A). At E14.5, Smek1 protein was detectable primarily in CP neurons (Figure 1B and S1B), while weak Smek1 expression was seen in some NPCs undergoing mitosis at the ventricle surface (Figure 1B, boxes). Interestingly, VZ neurons that migrate to the CP also expressed Smek1 protein (Figure 1B, arrows). In postnatal forebrain, Smek1 protein expression remained detectable in cortical layers I-IV (Figure S1C). Moreover, E14 cortices of Smek1-depleted mice (Smek1gt/gt) exhibited an increase in the number of Pax6-positive cells (an NPC marker) and a decrease in the number of Tbr1-positive cells (a marker of cortical neurons) as compared to E14 cortices of wild-type (Smek1+/+) mice (Figure 1C and S1D).
Smek1 regulates neuronal differentiation in the early phase of NPC differentiation
To assess Smek1’ function in neurogenesis, we employed an in vitro culture system using NPCs isolated from the E11.5 mouse forebrain neocortex. NPCs transduced with lentivirus expressing shRNA againstSmek1 or control shRNA under control of a doxycycline-inducible promoter (Figure S1E) were cultured in medium containing doxycycline for 6 days under differentiating conditions and then assessed for neurogenesis using TUJ1 (a marker of immature neurons) or MAP2 (a marker of mature neurons). The number of TUJ1- or MAP2-positive cells significantly decreased in Smek1 knockdown cultures compared to cultures expressing control shRNA (Figure 1D), indicating a neuronal differentiation defect. A decrease in number of neurons can be caused by a defect in NPC proliferation or neuronal apoptotic cell death. While no significant difference in the number of apoptotic cell death (as determined by TUNEL staining) was observed between control and Smek1 knockdown cells cultured under differentiation condition (data not shown),Smek1 knockdown NPCs grown under proliferation conditions underwent hyperproliferation (Figure S1F and G). We then asked whether Smek1 regulated the transition of NPCs from proliferative to differentiation states by knocking down Smek1 in NPCs prior to placing them in differentiating culture conditions. Western blotting of cells expressing Smek1 shRNA showed decreased levels of TUJ1 protein relative to controls by day 1 of culture (Figure S1H). At this time point, we found that the percentage of undifferentiated NPCs expressing both Nestin (an NPC marker) and Ki67 (a marker of proliferation) or Pax6 increased in cultures expressing Smek1 shRNA compared to control cultures, while the percentage of TUJ1-positive cells significantly decreased (Figure 1E and F). These findings suggest that Smek1 is required for neuronal differentiation and suppression of NPC proliferative capacity at an early phase of differentiation.
Smek1 recruits PP4c to promote neuronal differentiation
To determine Smek1 as a regulatory subunit of PP4 in neurogenesis, we asked whether Smek1 binds to the catalytic subunit PP4c in NPCs using co-immunoprecipitation. Western blot analysis revealed PP4c in Smek1 but not control immunoprecipitates, indicating that Smek1 physically interacts with PP4c. Such interactions did not change during differentiation (Figure 2A). To examine whether PP4c functions in neurogenesis, NPCs were exposed to lentivirus expressing PP4c or control shRNA and cultured as described in Figure 1D. PP4c knockdown led to changes similar to those accompanying Smek1 knockdown: relative to control cultures TUJ1 expression and the number of TUJ1-positive neurons decreased while Pax6-positive NPCs increased (Figure 2B and S2A and B). We next mapped Smek1 domains required for PP4c interaction. Smek contains four conserved domains: an N-terminal Ran-binding domain (RanBD), a domain of unknown function 625 (DUF625), an armadillo (Arm) repeat region, and a C-terminal nuclear localization sequence (NLS). We constructed a series of Flag-tagged deletion mutants, including Smek1ΔRanBD (lacking amino acid (aa) 2–100), ΔDUF625 (lacking aa 162-355), ΔArm (lacking 350-653), and ΔNLS (lacking aa 809-820) (Figure 2C, top) and introduced them or a wild-type construct into NPCs. PP4c was not be detected in anti-Flag immunoprecipitates from NPCs expressing Flag-Smek1ΔArm (Figure 2C, bottom) but was detected in cells expressing wild-type or other deletion mutants, suggesting that PP4c/Smek1 complex formation requires the Arm repeats. We also found that, while expression of wild-type Smek1 or corresponding ΔNLS mutant in cultures lacking endogenous Smek1 rescued the neuronal differentiation defect, the other mutants did not (Figure 2D and S2C and D). These results indicate that Smek1 regulates neuronal differentiation via its Arm repeats region through PP4c and suggest that both RanBD and DUF625 domains also participate in neurogenesis.
Smek1 binds to and mediates Par3 dephosphorylation
To identify PP4 substrates regulated by Smek1 in NPCs, we employed affinity purification to purify proteins interacting with Smek1. Mass spectrometry analysis identified potential Smek1-binding proteins, including Par3, Kinesin-like protein, coiled-coil domain-containing protein 30 (CCDC 30), heat shock protein 90 (HSP90), PKC lambda, and HDAC1 Figure S3A. Among these, Par3, an intrinsic regulator of neurogenesis, is a particularly attractive candidate (Bultje et al., 2009; Costa et al., 2008). Using an antibody that detects the major isoforms (180, 150, and 100 kDa) of Par3, Western blot analysis revealed the predominant expression of two isoforms, 180 and 100 kDa forms, in NPCs, and that only the 180 kDa Par3 was detectable in Smek1 immunoprecipitates (Figure 3A). To determine whether Smek1/Par3 binding was direct, we performed an in vitro pull-down assay using purified Flag-Smek1 and His-fused Par3 fragments, the latter containing the CR1 domain (aa 1-338), the PDZ domain (aa 343-733), the aPKC-BR domain (aa 711-1054), or the C-terminal coiled-coil region (aa 1055-1334) (Figure S3B). Western blot analysis revealed that Flag-Smek1 pulled down only the Par3 coiled-coil region (Figure 3B), indicating direct binding through that region. Moreover, Par3 was detected in Flag immunoprecipitates derived from NPCs transduced with lentivirus expressing Myc-Par3 plus lentivirus expressing Flag-Smek1 wild-type or Smek1ΔRanBD, Smek1ΔArm, or Smek1ΔNLS constructs but not from NPCs expressing Smek1ΔDUF625 (Figure 3C). This result indicates that Smek1 DUF625 domain is required for Smek1/Par3 interaction.
Smek1 interacts with Par3 and inhibits its function in neuronal differentiation
To assess potential dephosphorylation of Par3 by Smek1, we phosphorylayed Myc-Par3 protein in vitro by incubating it with an NPC lysate and 32P-ATP and then treated it with a complex containing Flag-Smek1 proteins (Figure 3D). 32P-labeling of Par3 was significantly decreased when Par3 protein was incubated with a complex containing wild-type Flag-Smek1 protein and PP4c (Figure 3D). By contrast, treatment with a Flag-Smek1ΔArm protein complex lacking PP4c binding significantly reduced Par3 dephosphorylation. Moreover, Western blot analysis of Par3 immunoprecipitates with an anti-phospho-serine/threonine antibody confirmed that Smek1 and PP4c regulate Par3 phosphorylation through serine/threonine residues (Figure S3C). Since the DUF625 and Arm repeats regions of Smek1 are required for binding to Par3 and PP4c respectively, we examined the Par3 phosphorylation state in NPCs expressing Flag-tagged wild-type or mutant Smek1 together with Myc-tagged Par3. Western blot analysis of Myc-Par3 immunoprecipitates using anti-phospho-serine/threonine antibody showed that overexpression of wild-type Smek1 or Smek1ΔNLS significantly decreased Par3 phosphorylation levels compared to controls, whereas overexpression of Smek1ΔRanBD, ΔDUF625, or ΔArm did not (Figure 3E). These results suggest that, in addition to the DUF625 and Arm, the RanBD domain of Smek1 participates in regulation of Par3 phosphorylation at serine/threonine residues.
Smek1 negatively regulates Par3 in neurogenesis
Next we asked whether Par3 is required for Smek1-mediated neurogenesis. To this end, we assessed the effect of Smek1 loss-or gain-of function on neuronal differentiation in the presence or absence of Par3. NPCs expressing either Smek1 shRNA or wild-type Smek1 were transduced with lentivirus expressing Par3 or control shRNA (Figure S3D). At day 1 after differentiation, in the presence of Par3, knockdown of Smek1 led to a decrease in the number of TUJ1-positive neurons and an increase in the number of Nestin/Ki67 double-positive NPCs, while overexpression of Smek1 had the opposite effect (Figure 3F and S3E). In the absence of Par3 by using shRNA, the number of TUJ1-positve cell was increased and the number of Nestin/Ki67 double positive NPCs was decreased. However, in these cultures knockdown or overexpression of Smek1 did not significantly alter the number of neurons or undifferentiated NPCs. In addition, we also observed increased expression of mRNAs encoding the Notch targets Hes1 and Hes5 in cells expressing Smek1 shRNA compared to control cells (Figure S3F). Moreover, analysis of Notch reporter gene activity revealed that wild-type Smek1 inhibited Notch signaling activity induced by Par3 overexpression, while Smek1ΔDUF625 did not (Figure S3G). Given that Par3 activates Notch signaling (Bultje et al., 2009), these results suggest that Smek1 acts upstream of Par3 to negatively regulate its activity in neurogenesis.
Par3 loss of function promotes neuronal differentiation (Costa et al., 2008), consistent with the effect seen following Smek1 overexpression (Figure 3F). To confirm that Smek1 promotes neurogenesis by suppressing Par3 function, we transduced NPCs with lentiviruses expressing Par3 alone or Par3 together with wild-type Smek1 or Smek1ΔDUF625, cultured them under differentiation conditions, and then neuronal differentiation was quantified by determining the percentage of TUJ1-positive and Nestin/Ki67 double-positive cells one day later. Par3 overexpression decreased the number of TUJ1-positive neurons and increased the number of Nestin/Ki67-positive undifferentiated NPCs compared with control cells (Figure 3G and S3H). As expected, wild-type Smek1 negated the effect of Par3 overexpression, as determined by comparing the percentage of TUJ1-positive and Nestin/Ki67 double-positive cells in cultures expressing both Smek1 and Par3 to cultures expressing Par3 alone. In comparison with wild-type Smek1, no significant change was seen in cultures transduced with Smek1ΔDUF625, which cannot bind Par3. These experiments further confirm that Smek1 negatively regulates Par3 in NPC differentiation.
Dynamic changes in Smek1 subcellular localization facilitate targeting of PP4 to Par3
Par3 localizes to the apical cortex of NPCs (Bultje et al., 2009), while Smek1 is predominantly nuclear (Figure 1A and C). To determine if changes in Smek1 subcellular localization occur in NPCs during neurogenesis, coronal sections from E11.5 forebrain were immunostained with anti-Smek1 and -α-tubulin (a cytoplasmic marker) antibodies. Smek1 co-localized with α-tubulin in cells on the ventricular surface (Figure 4A, arrows), indicating a cytoplasmic/cortical localization in mitotic NPCs. In mitotic cells, Par3 showed a similar localization (Figure S4A and B). Moreover, immunostaining of NPC cultures with anti-Smek1 and -α-tubulin antibodies showed that Smek1 undergoes dynamic changes in subcellular localization during mitosis. While Smek1 was nuclear in interphase and prophase cells, it showed a cytoplasmic/cortical localization from prometaphase to anaphase (Figure 4B and S4C). Metaphase and anaphase cells also showed Smek1 enrichment at spindle microtubules.
Smek1 regulates subcellular localization of PP4c but not Par3
The RanBD motif of the Dictyostelium discoideum Smek homolog is reportedly critical for its cytoplasmic/cortical localization (Mendoza et al., 2005). To test whether this was the case for mammalian Smek1, Smek1-depleted NPCs were transduced with constructs encoding Flag-tagged wild-type Smek1 or its deletion mutants and immunostained with anti-Flag and anti-phospho-histone H3 (a marker of mitosis and chromatin condensation). Consistent with results reported in Dictyostelium discoideum, the Smek1ΔRanBD mutant failed to localize to the cytoplasm/spindle during mitosis but rather localized in the nucleus and remained there in interphase (Figure 4C). The subcellular localization of other mutants tested resembled that of wild-type Smek1, with the exception of Smek1ΔNLS, which was expressed in both the nucleus and cytoplasm of interphase cells. Smek1ΔRanBD contains domains that can bind Par3 and PP4c, as shown by immunoprecipitation (Figure 2C and and3C).3C). We thus asked whether ectopic expression of Smek1ΔRanBD promoted mislocalization of Par3 and PP4c during mitosis. When we expressed Smek1ΔRanBD ectopically in Smek1-depleted NPCs, cytoplasmic/cortical Par3 remained unchanged while Smek1ΔRanBD was nuclear (Figure S4D). In addition, no difference in localization of Par3 between Smek1-depleted and wild-type Smek1 re-expressing cells was observed, suggesting that Smek1 does not alter Par3 localization. To evaluate PP4c subcellular localization, chromosome-associated and cytosolic protein fractions were isolated from M phase-synchronized NPCs and compared by Western analysis using indicated antibodies (Figure 4D). Interestingly, PP4c protein levels increased in the chromosomal fraction from cells expressing Flag-Smek1ΔRanBD compared to control cells or wild-type Flag-Smek1, while in the cytosolic fraction the level of PP4c protein decreased, indicating altered localization of cytoplasmic PP4c to the nucleus. Taken together, these results demonstrate that PP4c subcellular localization depends on Smek1 localization during mitosis and suggest that cytoplasmic/cortical localization of Smek1 targets PP4 to Par3.
DISCUSSION
Neural stem and progenitor cells have been suggested as potential therapeutics for neurodegenerative disorders. However, understanding molecular and cellular mechanisms underlying their differentiation is a prerequisite to manipulating stem cell behavior. We show that Smek1, an evolutionarily conserved regulatory subunit of PP4, regulates neuronal differentiation and reveal an unreported function of PP4 in mammalian neurogenesis. Moreover, identification of Par3 as a novel Smek1-interacting protein and characterization of its conserved domains reveals a molecular mechanism by which Smek1 targets PP4 to Par3 during mitosis and negatively regulates Par3 function in neurogenesis.
In this study we identify Par3 as a PP4 substrate. We propose that Smek1, through its DUF625 domain, binds directly to the Par3 C-terminus. In NPCs Par3 is primarily cytoplasmic in interphase and mitosis. Thus, nuclear export of Smek1 to the cytoplasm is required for its interaction with Par3. We show dynamic changes in Smek1 subcellular localization in NPCs. While Smek1 localizes exclusively to the nucleus in interphase, during mitosis it becomes cytoplasmic. The RanBD of several proteins reportedly recognizes GTP-bound Ran (RanGTP), which directs assembly of spindle microtubules allowing chromosomal segregation and cytokinesis in mitosis (Carazo-Salas et al., 2001). Smek1 enrichment at spindle microtubules in metaphase and anaphase cells suggests that its RanBD may function in a RanGTP-dependent pathway during mitosis. Notably, nuclear export of Smek1 to the cytoplasm was observed from prometaphase cells when microtubules invade the nuclear space, and deletion of the Smek1 RanBD abolished this effect, as seen by nuclear localization. Thus our data suggest that Smek1 subcellular localization is regulated through the RanBD and that this activity may depend on microtubule dynamics functioning in a Ran-dependent pathway.
Most Smek homologs physically interact with the catalytic subunit PP4c (Gingras et al., 2005; Chowdhury et al., 2008), suggesting that the PP4 complex is evolutionarily conserved. We show that PP4c recognizes the Arm repeats region of Smek1 and its subcellular localization depends on Smek1 localization. Thus, nuclear export of Smek1 during mitosis facilitates dephosphorylation of Par3. This idea is supported by our observation that, while expression of Smek1 induced Par3 dephosphorylation in NPCs, expression of Smek1 mutants lacking RanBD and Arm repeats region did not. Interestingly, studies of Drosophila neuroblasts previously revealed that cell fate specification is tightly linked with phosphorylation status of bazooka protein (Betschinger et al., 2003; Krahn et al., 2009). However, it is now yet clear whether Par3 dephosphorylation directly regulates NPC neurogenesis. Although we could not identify specific phosphorylation sites targeted by PP4, our data defines three conserved domains of Smek1, namely RanBD, DUF625, and Arm repeats, necessary to target PP4 to its substrate Par3 and provides insight into the molecular mechanism by Smek1 to regulate PP4 function in NPCs.
We here show that Smek1 suppresses Par3, a negative regulator of neuronal differentiation. Par3 acts upstream of Notch signaling (Bultje et al., 2009), which critically regulates cell fate decision of NPCs in cortical development (Gaiano and Fishell, 2002). Notch gain-of-function activity inhibits neuronal differentiation (Nye et al., 1994), an effect similar to Smek1 loss-of-function. Moreover, Smek1 inhibits Par3-induced Notch reporter gene activity. Although it remains unclear whether Smek1 inhibits Par3’s ability to activate Notch signaling during mitosis, ensuring a neuronal fate, our data demonstrate Smek1 as a negative regulator of Par3 in regulating neuronal differentiation and suggest a novel role for PP4 in mammalian neurogenesis.
Acknowledgments We thank the USC Transgenic Core Facility for generating mutant mice. This research is funded by a NIH grant to W.L (5R01NS067213) and an NRF grant (NRF-2011-35B-E00015) to J.L.
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NEW YORK (GenomeWeb News) – A quartet of Boston-area research centers including Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Boston Children’s Hospital, and the Broad Institute have teamed to create a new clinical cancer genomics center that will be headquartered at Dana-Farber.
Dana-Farber said today that the new Joint Center for Cancer Precision Medicine will harness a wide range of scientific resources and clinical capabilities from the partners to treat cancer patients and feed treatment data into research programs. The multiple capabilities these partners will share and use in the new center include DNA sequencing and other tumor molecular profiling tools, pathology, radiology, surgery, computational interpretation, and tumor modeling systems, they said.
“The center is creating a new capability to use these huge resources in sequencing and pathology and making sure the data gets to caregivers to help customize cancer treatment,” Dana-Farber President Edward Benz said in a statement.
A core part of the center will be a program to obtain and characterize biopsies from patients during treatment by looking at the tumors’ DNA, RNA, and proteins.
The center also will create a computational biology working group that will spread across Dana-Farber, Broad, and Brigham and Women’s Hospital and will include biologists, bioinformaticians, and software designers to develop algorithms aimed at interpreting genome sequencing data.
The partners also plan to support a translational innovation lab that will pursue studies on actionable cancer mutations and drug resistance, as well as preclinical studies of targeted drug combinations. In addition, they will work with members of the Profile cancer genetics research study, a project already launched by Dana-Farber and Brigham and Women’s that is focused on analyzing tumor DNA and creating a database of relevant mutations.
The CanSeq study, a whole-exome sequencing effort involving Dana-Farber, Brigham and Women’s and Broad investigators, will become “an integral part of the new center,” as researchers plan to study the value of whole-exome sequencing in cancer treatment, Dana-Farber said. Currently, the CanSeq partners are sequencing the whole exomes of 50 lung and colon cancer patients as part of a pilot phase.
“This center will allow us to be optimally positioned to answer the big questions in cancer genetics, especially as they affect clinical decision-making,” said Levi Garraway, an associate professor at Dana-Farber and the new center’s director, as well as head of the CanSeq study.
“We seek to understand which genetic and other molecular alterations predict how tumors will respond to targeted drugs, why some patients become resistant to drugs, and what that means about the treatments that should be tried next,” he added.
Discovery on Target: Industry’s Preeminent Event on Novel Drug Targets
Reporter: Aviva Lev-Ari, PhD, RN
ABOUT THIS CONFERENCE
Cambridge Healthtech Institute’s tenth annual conference on Functional Genomics Screening Strategies will cover the latest in the use of RNA interference (RNAi) screens, combination (RNAi and small molecule) screens, chemical genomics and phenotypic screens, for identifying and validating new drug targets and exploring unknown cellular pathways. The first half of the conference will focus on the design and use of RNAi screens, while the second half will explore the use of chemical genomics screens, microRNA (miRNA) and long non-coding RNA (LncRNA) screens and the transition into advanced cellular models such as, 3D cell cultures, co-cultures and stem cells that will launch the next-generation of functional screens. Screening experts from pharma/biotech as well as from academic and government labs will share their experiences leveraging the utility of such diverse screening platforms and models for a wide range of applications.
SUGGESTED EVENT PACKAGE:
September 23: Setting Up Effective RNAi Screens: Getting From Design to Data Short Course September 24 – 25: Functional Genomic Screening Strategies Conference Part One September 25: Setting Up Effective Functional Screens Using 3D Cell Cultures Dinner Short Course September 25 – 26: Functional Genomic Screening Strategies Conference Part Two
WHERE AND HOW TO APPLY SIRNA AND SHRNA SCREENS
Comparative Analysis of Arrayed RNAi Screening Performance of siRNA versus shRNA at Genome-Scale
Swimming in the Deep End – Sources Leading to a False Sense of Security in RNAi Screen Data
Scott Martin, Ph.D., Team Leader, RNAi Screening, NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Institutes for Health
Target Identification and Validation of Novel Ion Channels in Cancer
Alex Gaither, Ph.D., Research Investigator II, Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research
Cell-Based Functional Profiling of Lipid-Traits and Cardiovascular Disease
Heiko Runz, M.D., Group Leader, Molecular Metabolic Disease Unit, Institute of Human Genetics; Group Leader, University of Heidelberg
Use of Functional Genomics to Identify Patients at High Risk for Recurrence of Hepatitis C Following Liver Transplantation
Robert Carithers, M.D., Professor of Medicine, Director, Liver Care Line; Medical Director, Liver Transplant Program, University of Washington Medical Center
Pooled RNAi Genetic Screening to Identify Functional Genes and Novel Drug Targets
Paul Diehl, Director, Business Development, Cellecta, Inc.
TECHNOLOGY PANEL: Tools for Next-Generation Functional Genomics Screens
Moderator: Christophe Echeverri, Ph.D., CEO & CSO, Cenix BioScience USA, Inc.
This panel will bring together 4-5 technical experts from leading technology and service companies to discuss screening trends and improvements in assay platforms and reagents that users can expect to see in the near future.
Curators: Aviva Lev-Ari, PhD, RN and Larry Bernstein, MD, FACP
The essence of the message is summarized by Larry Bernstein, MD, FACP, as follows:
[1] we employ a massively parallel reporter assay (MPRA) to measure the transcriptional levels induced by 145bp DNA segments centered on evolutionarily-conserved regulatory motif instances and found in enhancer chromatin states
[2] We find statistically robust evidence that (1) scrambling, removing, or disrupting the predicted activator motifs abolishes enhancer function, while silent or motif-improving changes maintain enhancer activity; (2) evolutionary conservation, nucleosome exclusion, binding of other factors, and strength of the motif match are all associated with wild-type enhancer activity; (3) scrambling repressor motifs leads to aberrant reporter expression in cell lines where the enhancers are usually not active.
[3] Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale experimental manipulation, and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to generate and test predictive models of gene expression.
Manolis Kellis and co-authors from the Massachusetts Institute of Technology and the Broad Institute describe a massively parallel reporter assay that they used to systematically study regulatory motifs falling within thousands of predicted enhancer sequences in the human genome. Using this assay, they examined 2,104 potential enhancers in two human cell lines, along with another 3,314 engineered enhancer variants. “Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale experimental manipulation,” they write, “and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to generate and test predictive models of gene expression.”
Genome-wide chromatin maps have permitted the systematic mapping of putative regulatory elements across multiple human cell types, revealing tens of thousands of candidate distal enhancer regions. However, until recently, their experimental dissection by directed regulatory motif disruption has remained unfeasible at the genome scale, due to the technological lag in large-scale DNA synthesis. Here, we employ a massively parallel reporter assay (MPRA) to measure the transcriptional levels induced by 145bp DNA segments centered on evolutionarily-conserved regulatory motif instances and found in enhancer chromatin states. We select five predicted activators (HNF1, HNF4, FOXA, GATA, NFE2L2) and two predicted repressors (GFI1, ZFP161) and measure reporter expression in erythroleukemia (K562) and liver carcinoma (HepG2) cell lines. We test 2,104 wild-type sequences and an additional 3,314 engineered enhancer variants containing targeted motif disruptions, each using 10 barcode tags in two cell lines and 2 replicates. The resulting data strongly confirm the enhancer activity and cell type specificity of enhancer chromatin states, the ability of 145bp segments to recapitulate both, the necessary role of regulatory motifs in enhancer function, and the complementary roles of activator and repressor motifs. We find statistically robust evidence that (1) scrambling, removing, or disrupting the predicted activator motifs abolishes enhancer function, while silent or motif-improving changes maintain enhancer activity; (2) evolutionary conservation, nucleosome exclusion, binding of other factors, and strength of the motif match are all associated with wild-type enhancer activity; (3) scrambling repressor motifs leads to aberrant reporter expression in cell lines where the enhancers are usually not active. Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale experimental manipulation, and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to generate and test predictive models of gene expression.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by-nc/3.0/.
Genomics and Ethics: DNA Fragments are Products of Nature or Patentable Genes?
Curator: Aviva Lev-Ari, PhD, RN
UPDATED 6/17/2013 – OPINIONS ON COURT DECISION of 6/13/2013
Experts say court’s decision on human gene patents is a win-win
Jun 16, 2013
Jun 16, 2013 (St. Louis Post-Dispatch – McClatchy-Tribune Information Services via COMTEX News Network) — The Supreme Court ruling Thursday that naturally occurring human genes cannot be patented effectively ended the monopoly that Utah-based Myriad Genetics had on breast and ovarian cancer tests.
The news was hailed as a victory by health advocates and medical researchers, who can now not only access the genes at issue — the BRCA1 and BRCA2 — but all other patented human genes without infringement. In the wake of the decision, several other testing companies, including Quest Diagnostics, announced it would perform the tests — and at far cheaper prices than Myriad’s.
The court’s unanimous ruling, however, was mixed. It said that naturally occurring DNA could not be patented, but synthetic DNA can still be, giving patent protection advocates and Myriad a victory, too.The decision also means that methods of isolating genes still qualify for patent protection.
The Post-Dispatch interviewed experts from a broad range of fields, from medicine to law, about the court’s ruling.
Here’s what they had to say about what was at stake and what the decision could mean.
Christopher Mason
Professor of physiology, biophysics and computational biomedicine, and author of a study showing that 41 percent of the human genome is covered by patents, Cornell University
I’d say this represents a great win for genetic liberty, both for patients and for doctors. The American Medical Association said it was a big win for patients, and I couldn’t agree more — especially for breast and ovarian cancer, but for all types of cancer. This is an important cancer gene and now it’s open for study to everyone.
(Myriad) didn’t just own a test or a method, they owned anyone’s DNA as soon as it was isolated. They didn’t say we patented a series of letters, they said we patent anything that remotely looks like that, which the court correctly said is not patentable.
It would have been great to have both the patents (on natural and synthetic DNA), but of the two this is the most restrictive one — 99.9 percent of testing is done on DNA not cDNA.
Plenty of companies aren’t scared anymore. This is going to open the floodgates on new research and ideas.
Dr. Julie Margenthaler
Associate professor of surgery and breast cancer specialist, Siteman Cancer Center
This ruling has important implications for physician scientists actively engaged in genetic research. We are on the brink of significant strides in our understanding of the genetic links to many diseases.
For those of us who care for cancer patients, personalized cancer care hinges on the ability to genetically examine the pathways that result in a normal cell becoming a malignant cell. Because some companies held patents to pieces of the genome involved when whole genome sequencing is performed, there was at least some concern over patent infringement. With this ruling, we can continue to move our research forward and benefit the lives of our current and future patients.
Michael Watson
Executive director, American College of Medical Genetics and Genomics (plaintiffs in the case), and former professor of pediatrics at Washington University
It has enormous implications for labs and the public, certainly for breast cancer and for many other cancers. Since the case was settled (Thursday), at least four labs have put the test online. Prices are about half of Myriad’s — $3,500 down to $2,000 overnight.
It’s a win-win for everybody. It used to be when you had the tests done by Myriad, you couldn’t get that test confirmed by anyone else. Now the public can confirm the test and get second opinions, and that has a lot of value for patients. And I think it’ll open up the research.
There are two aspects of this that still remain open. Because 4,000 to 5,000 genes have patents on them, many people signed licensing agreements to use the gene. One of the questions is about the contract they signed. They will probably be able to challenge their contract now.
Nathan Lakey President and CEO, Orion Genomics
I think the ruling is positive because it removes a cloud of uncertainty as to where the Supreme Court stood on patents relating to gene sequences. I appreciate the thoughtfulness that went into the ruling. Justice Thomas adds a section that talks about what the ruling did not address that’s interesting. He emphasizes that method patents, or patents covering gene sequences that apply knowledge of those sequences, are patentable. I think this is what the justices sought to do, to not limit science and to not limit innovation and improvements in patient care. I think they do a markedly good job laying out the framework by which the business of science needs to consider the issue going forward as we all seek to lower the cost of care and improve outcomes.
We’re thrilled because our patents have been crafted primarily as method patents that involve naturally occurring gene sequences, and at the same time we add on to that a novel method that was not known and is quite valuable. We have biomarkers that we believe will be able to predict the risk of an individual getting colon cancer in the future, not unlike the Myriad test, but this is for colon cancer. We feel that our path forward is actually more clear and more positive given the clear line that the Supreme Court drew around what is and what isn’t patentable.
Janet S. Hendrickson
Patent attorney, specializing in chemical, pharmaceutical and food science companies, Senniger Powers law firm
They split it down the middle, and it seems to be, when looking at the commentary, that most people agree with that. They didn’t preclude the patenting of everything related to DNA, just natural DNA.
There are so many considerations and it’s hard to know what ramifications there are going to be, and what might be the best policy. It does mean that for companies that have these claims on natural DNA in their portfolio, they need to make sure they have the other range of claims for the cDNA (synthetic DNA). For companies that have past patents, it’s going to figure into those claims for those natural DNA products.
So it’s hard to tell whether it has broader implications for other things, that when you take them out of their natural milieu we thought were patentable.
Kevin Emerson Collins Professor, Washington University School of Law and patent law expert
This is going to mean one thing for patent lawyers and another thing for biotech companies. For patent lawyers, we now have a new source of business. The court hasn’t given us precise guidelines that say exactly when in other situations do we pass from something being a product of nature to a patentable invention. That’s a new frontier that patent lawyers are going to have to advise companies on.
For biotech companies it’s going to mean they pay patent lawyers a little more. Although the Myriad Genetics ruling deals with DNA, it would seem from the language of the opinion that the ruling should also apply to nongenetic, naturally occurring materials, but exactly how is yet to be determined.
A historical example that predates the Myriad controversy is the debate over the patentability of insulin in the early 20th century. A very famous lower court opinion held that isolated and purified human insulin was patentable so long as it became isolated insulin with impurities removed and took on new commercial value. I bet that case might well come out differently under the Myriad Genetics ruling. The insulin question is moot; that patent has expired. Similarly there a number of other therapeutics which are components that nature already makes that are isolated in a way they can be used in medicine but not in their natural state. Those are the kinds of things we’re going to have to grapple with.
Josh Newby-Harpole Founder, Theresa Harpole Foundation for Metastatic Breast Cancer in Alton
We have a foundation we started this year in honor of my mom. She was diagnosed over seven years ago with stage zero breast cancer. They did genetic testing and found out she had the BRCA gene. In 2010 she got diagnosed with metastatic breast cancer after she had a lump in her neck and it had spread to her bones. I needed to get tested at that point. I had testing done in Chicago and found out that I had the BRCA gene. As a male I’m lucky she had a son and not a daughter. My mom has been on different courses of treatment, and I monitor my health as well as I can, because I have a higher risk for certain kinds of cancer such as prostate and skin cancer and a higher than 3 percent chance of breast cancer.
The cost was probably over $2,000 to have the test done, and I paid close to $1,000 for it. We’re very excited about the Supreme Court ruling. I think a lot of people are hesitant to get the test done because of the cost. It’s exciting because it means possibilities. More people are going to be motivated to do research in labs to try to find a cure. Maybe they can come up with better treatment options for women because some of them will find out they have the gene and they don’t have evidence of disease. It’s something that is really getting a lot of attention right now, and the population is maybe not as aware about things like BRCA and metastatic breast cancer.
Yvette Liebesman Assistant professor of law, St. Louis University
It’s very good for research and in fact it’s very good for health care in the sense that already today a competitor for Myriad said they would run the same test for thousands less. Already we’re seeing a good thing happening that more women are going to be able to be tested for this gene. Now we’re talking about more women being aware of their health risks. Now a company that wants to develop a drug isn’t going to have to go through Myriad to isolate this gene in order to test drugs for breast cancer.
If Myriad won this case it would be like saying while a tree is made by nature, if I find a way to pick the leaves off it, the leaf is my patented product. Myriad did win in one sense, that there is a form of DNA not found in nature that is patentable. This is very logical. I think that like with most things, the people who are doomsayers will say it’s not going to have as great of an impact. The idea that now this opens up the ability to develop treatments is going to be huge.
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www.stltoday.com Distributed by MCT Information Services
The Supreme Court ruled unanimously Thursday that human genes cannot be patented, a decision that could shape the future of medical and genetic research and have profound effects on pharmaceuticals and agriculture.The ruling was a split decision for Myriad Genetics Inc., which holds patents on genes that have been linked to breast and ovarian cancer.
Justice Clarence Thomas, writing for the court, said that merely isolating those specific genes — called BRCA1 and BRCA2 — was not worthy of a patent.
“Myriad found the location of the BRCA1 and BRCA2 genes, but that discovery, by itself, does not render the BRCA genes . . . patent eligible,” Thomas wrote.On the other hand, Thomas wrote, Myriad’s creation of a synthetic form of DNA — called cDNA — based on its discovery does deserve patent protection.“The lab technician creates something new when cDNA is made,” Thomas wrote.Responding to the decision, Myriad focused on the favorable cDNA ruling. “We believe the court appropriately upheld our claims on cDNA, and underscored the patent eligibility of our method claims, ensuring strong intellectual property protection for our BRACAnalysis test moving forward,” said Peter D. Meldrum, company president and chief executive. “More than 250,000 patients rely upon our BRACAnalysis test annually, and we remain focused on saving and improving peoples’ lives and lowering overall health-care costs.”DNA research is a vital component of personalized medicine. The challenge to Myriad’s patents came from scientists and doctors who said that allowing patents on genes inflated the cost of testing and hindered research.
The American Civil Liberties Union praised the high court’s ruling as a victory. “Today, the court struck down a major barrier to patient care and medical innovation,” said Sandra Park of the ACLU, which represented the groups that brought the challenge. “Because of this ruling, patients will have greater access to genetic testing, and scientists can engage in research on these genes without fear of being sued.”
The test that Myriad offers for determining whether a woman contains the genetic mutation that heightens her chance of cancer has received much attention lately after actress Angelina Jolie wrote about it in a letter to the editor to the New York Times. In the letter, Jolie revealed that she had a double mastectomy because the test showed she carried the defective gene.
NOTE: Where it is feasible, a syllabus (headnote) will be released, as is being done in connection with this case, at the time the opinion is issued.The syllabus constitutes no part of the opinion of the Court but has been prepared by the Reporter of Decisions for the convenience of the reader. See United States v. Detroit Timber & Lumber Co., 200 U. S. 321, 337.
CERTIORARI TO THE UNITED STATES COURT OF APPEALS FOR THE FEDERAL CIRCUIT
No. 12–398. Argued April 15, 2013—Decided June 13, 2013
Each human gene is encoded as deoxyribonucleic acid (DNA), which takes the shape of a “double helix.” Each “cross-bar” in that helix consists of two chemically joined nucleotides. Sequences of DNA nucleotides contain the information necessary to create strings of amino acids used to build proteins in the body. The nucleotides that code for amino acids are “exons,” and those that do not are “introns.” Scientists can extract DNA from cells to isolate specific segments for study. They can also synthetically create exons-only strands of nucleotides known as composite DNA (cDNA). cDNA contains only the exons that occur in DNA, omitting the intervening introns. Respondent Myriad Genetics, Inc. (Myriad), obtained several patents after discovering the precise location and sequence of the BRCA1 and BRCA2 genes, mutations of which can dramatically increase the risk of breast and ovarian cancer. This knowledge allowed Myriad to determine the genes’ typical nucleotide sequence, which, in turn, enabled it to develop medical tests useful for detecting mutations in these genes in a particular patient to assess the patient’s cancer risk. If valid, Myriad’s patents would give it the exclusiveright to isolate an individual’s BRCA1 and BRCA2 genes, and would give Myriad the exclusive right to synthetically create BRCA cDNA. Petitioners filed suit, seeking a declaration that Myriad’s patents areinvalid under 35 U. S. C. §101. As relevant here, the District Court granted summary judgment to petitioners, concluding that Myriad’s claims were invalid because they covered products of nature. The Federal Circuit initially reversed, but on remand in light of Mayo Collaborative Services v. Prometheus Laboratories, Inc., 566 U. S. ___, the Circuit found both isolated DNA and cDNA patent eligible. 2 ASSOCIATION FOR MOLECULAR PATHOLOGY v. MYRIAD GENETICS, INC. Syllabus
Held: A naturally occurring DNA segment is a product of nature and not patent eligible merely because it has been isolated, but cDNA is patent eligible because it is not naturally occurring. Pp. 10–18.
(a) The Patent Act permits patents to be issued to “[w]hoever invents or discovers any new and useful . . . composition of matter,” §101, but “laws of nature, natural phenomena, and abstract ideas”“ ‘are basic tools of scientific and technological work’ ” that lie beyond the domain of patent protection, Mayo, supra, at ___. The rule against patents on naturally occurring things has limits, however. Patent protection strikes a delicate balance between creating “incentives that lead to creation, invention, and discovery” and “imped[ing] the flow of information that might permit, indeed spur, invention.” Id., at ___. This standard is used to determine whether Myriad’s patents claim a “new and useful . . . composition of matter,” §101, or claim naturally occurring phenomena. Pp. 10–11.
(b) Myriad’s DNA claim falls within the law of nature exception.Myriad’s principal contribution was uncovering the precise location and genetic sequence of the BRCA1 and BRCA2 genes. Diamond v. Chakrabarty, 447 U. S. 303, is central to the patent-eligibility inquiry whether such action was new “with markedly different characteristics from any found in nature,” id., at 310. Myriad did not create or alter either the genetic information encoded in the BCRA1 and BCRA2 genes or the genetic structure of the DNA. It found an important and useful gene, but ground breaking, innovative, or even brilliant discovery does not by itself satisfy the §101 inquiry. See Funk Brothers Seed Co. v. Kalo Inoculant Co., 333 U. S. 127. Finding the location of the BRCA1 and BRCA2 genes does not render the genes patent eligible “new . . . composition[s] of matter,” §101. Myriad’s patent descriptions highlight the problem with its claims: They detail the extensive process of discovery, but extensive effort alone isinsufficient to satisfy §101’s demands. Myriad’s claims are not saved by the fact that isolating DNA from the human genome severs the chemical bonds that bind gene molecules together. The claims are not expressed in terms of chemical composition, nor do they rely on the chemical changes resulting from the isolation of a particular DNA section. Instead, they focus on the genetic information encoded in the BRCA1 and BRCA2 genes. Finally, Myriad argues that the Patent and Trademark Office’s past practice of awarding gene patents is entitled to deference, citing J. E. M. Ag Supply, Inc. v. Pioneer Hi-Bred Int’l, Inc., 534 U. S. 124, a case where Congress had endorsed a PTO practice in subsequent legislation. There has been no such endorsement here, and the United States argued in the Federal Circuit and in this Court that isolated DNA was not patent eligible under §101. Pp. 12–16.
3 Cite as: 569 U. S. ____ (2013)
Syllabus
(c) cDNA is not a “product of nature,” so it is patent eligible under§101. cDNA does not present the same obstacles to patentability as naturally occurring, isolated DNA segments. Its creation results in an exons-only molecule, which is not naturally occurring. Its order of the exons may be dictated by nature, but the lab technician unquestionably creates something new when introns are removed from a DNA sequence to make cDNA. Pp. 16–17.
(d) This case, it is important to note, does not involve method claims, patents on new applications of knowledge about the BRCA1 and BRCA2 genes, or the patentability of DNA in which the order of the naturally occurring nucleotides has been altered. Pp. 17–18.
689 F. 3d 1303, affirmed in part and reversed in part.
THOMAS, J., delivered the opinion of the Court, in which ROBERTS, C. J., and KENNEDY, GINSBURG, BREYER, ALITO, SOTOMAYOR, and KAGAN, JJ., joined, and in which SCALIA, J., joined in part. SCALIA, J., filed an opinion concurring in part and concurring in the judgment.
1 Cite as: 569 U. S. ____ (2013) Opinion of SCALIA, J.
SUPREME COURT OF THE UNITED STATES
No. 12–398
ASSOCIATION FOR MOLECULAR PATHOLOGY, ET AL., PETITIONERS v. MYRIAD GENETICS, INC., ET AL.
ON WRIT OF CERTIORARI TO THE UNITED STATES COURT OF APPEALS FOR THE FEDERAL CIRCUIT
[June 13, 2013]
JUSTICE SCALIA, concurring in part and concurring in the judgment.
I join the judgment of the Court, and all of its opinion except Part I–A and some portions of the rest of the opinion going into fine details of molecular biology. I am un-able to affirm those details on my own knowledge or even my own belief. It suffices for me to affirm, having studied the opinions below and the expert briefs presented here, that the portion of DNA isolated from its natural state sought to be patented is identical to that portion of the DNA in its natural state; and that complementary DNA (cDNA) is a synthetic creation not normally present in nature.
Evolution of the case ASSOCIATION FOR MOLECULAR PATHOLOGY ET AL. v. MYRIAD GENETICS, INC., ET AL. priot to 6/13/2013 Supreme Court decision
Curator: Aviva Lev-Ari, PhD, RN
In an amicus brief, the Broad Institute‘s Eric Lander shares his personal view of the ongoing gene patenting case between Myriad Genetics and the American Civil Liberties Union, saying that isolated DNA fragments are products of Nature.
The central issue of the case revolves around Myriad’s patents on the BRCA1 and BRCA2 genes. In a mixed ruling, the federal appeals court found that while some of the company’s methods patents may not be patentable, its BRCA1 and BRCA2 gene patents, as they concern isolated DNA fragments, are patentable items as human intervention is needed to isolate DNA.
Lander argues that that is not true, though, as the Boston Globe points out, his brief was not filed in support of either side. Isolated DNA, he says, happens all the time in nature. “It is well-accepted in the scientific community that
(a) chromosomes are constantly being broken into DNA fragments by natural biological processes that break the covalent bonds within DNA chains;
(b) these DNA fragments can be routinely found in the human body … and
(c) these fragments cover the entire human genome and, in particular, include many of the DNA fragments claimed by Myriad’s patents,” the brief says.
The US Supreme Court announced in December that it will re-hear the Myriad gene patenting case.
Late last year, the nation’s highest court said it would consider a legal challenge to patents that biotechnology company Myriad Genetics holds on breast cancer genes. Now, Eric Lander, head of the Broad Institute in Cambridge, has filed an amicus brief that he says reflects his personal opinion. Utah-based Myriad, Lander argues, has patented products of nature, and its patents are an “insurmountable barrier” to studying DNA, with serious repercussions for medical progress.
1. THE FEDERAL CIRCUIT INCORRECTLY ASSUMED, WITHOUT CITING SCIENTIFIC EVIDENCE, THAT ISOLATED DNA FRAGMENTS OF THE HUMAN GENOME DO NOT OCCUR IN NATURE, WHEN IT IS WELL-ACCEPTED IN THE SCIENTIFIC COMMUNITY THAT THEY DO
2. MYRIAD’S COMPOSITION-OF-MATTER CLAIMS ON ISOLATED FRAGMENTS OF THE GENOMIC DNA ARE INCONSISTENT WITH THIS COURT’S SECTION 101 JURISPRUDENCE BECAUSE THEY (1) ARE DIRECTED TO PREEXISTING PRODUCTS OF NATURE (2) EXCLUDE OTHERS FROM OBSERVING, CHARACTERIZING OR ANALYZING THESE PRODUCTS OF NATURE BY ANY MEANS WHATSOEVER; AND (3) CREATE AN INSURMOUNTABLE BARRIER TO SCIENTIFIC PROGRESS AND TECHNOLOGICAL INNOVATION CONCERNING THESE PRODUCTS OF NATURE
3. A NARROWLY CRAFTED DECISION BY THIS COURT WOULD NOT UNDERMINE THE BIOTECHNOLOGY INDUSTRY AND INSTEAD WOULD FOSTER INNOVATION
CONCLUSION
It is well-accepted in the scientific community that isolated DNA fragments of the human genome – including isolated DNA fragments of the BRCA1 and BRCA2 genes – are found routinely in th human body and are thus patent-ineligible products of Nature. The biotechnology industry would not be substantially affected by a narrowly crafted decision here holding that
1) fragments of human genome DNA are patent-ineligible where the claimed molecules themselves are routinely found in Nature and where the process for purification or synthesis of such molecules iS routine and
The Supreme Court should be mindful of naturally derived products other than nucleic acids when deciding Myriad
The following contribution to our gene patenting symposium come from Susan McBee and Bryan Jones. Ms. McBee is the Chair of the Life Sciences Intellectual Property Team for Baker, Donelson, Bearman, Caldwell, and Berkowitz, P.C. Bryan Jones is a registered patent attorney in the Washington D.C. office of Baker, Donelson, Bearman, Caldwell, and Berkowitz, P.C.
In April, the Supreme Court will hear oral argument in Association for Molecular Pathology v. Myriad, ostensibly on the question whether so-called “gene patents” satisfy 35 U.S.C. § 101. However, Myriad is about more than whether “genes” can be patented. It is about what types of activities justify patent protection. Does one need to create something that is unlike anything else that has ever existed in order to justify a patent? Or is it enough to discover something that was previously unknown, remove it from its natural environment, and show that it has a practical application?
This is a critical question to the biotechnology industry, because many biotechnological products are not novel chemical structures, but naturally occurring products. Between 1981 and 2006, approximately forty percent of all pharmaceuticals approved for use by the FDA were a biologic, natural product, or derived from a natural product. Moreover, for start-up biotechnology companies, patents covering such products are incredibly important, “as they are often the most crucial asset they own in a sector that is extremely research-intensive and with low imitation costs.” Strong and enforceable patents to these core products therefore are vitally important to the healthy development of the biotechnology industry.
Before the Myriad case, the Court has not had an opportunity to consider the patentability of such products. Therefore, this case has the potential to have an enormous impact on the viability of the business model in this industry.
In Myriad, Judge Lourie and Judge Moore both found “isolated” nucleic acids to be patentable, but for different reasons. Judge Lourie was convinced that isolated nucleic acids are patentable because isolation “breaks covalent bonds” relative to the longer native nucleic acid, thereby resulting in a new chemical entity. Judge Moore reasoned that, if analyzed on a blank slate, she would require the product to have a “substantial new utility” relative to its natural function in order to satisfy 35 U.S.C. § 101. While we agree that the generation of a novel chemical entity or demonstration of a new utility would be sufficient to satisfy 35 U.S.C. § 101, we do not believe these to be necessary requirements.
Consider, for example, Taq polymerase. The inclusion of Taq into a process called polymerase chain reaction (PCR) has often been credited as being the single most important technological advance to the modern biotechnology industry. PCR uses repeated cycles of increasing and decreasing temperatures in the presence of a polymerase to amplify a target nucleic acid. In the original iteration of PCR, new polymerase enzyme had to be added to the reaction mixture after each heat cycle, because the high temperature permanently deactivated the enzyme. Taq, however, is heat stable and thus does not lose activity when subjected to high temperatures. Because of this stability, Taq only needs to be added to a PCR reaction mixture once, thus greatly reducing the costs and the time of performing the process, and permitting easy automation. Clearly, then, the identification and characterization of this enzyme is a significant technological advance, from which the public obtains a significant benefit. Yet the properties of Taq thatmake it so attractive for PCR are a consequence of its structure and function in the natural world. Taq is naturally produced by Thermus aquaticus, a bacterium that is naturally found in hot springs. Therefore, in nature, just like in PCR, Taq functions as a thermostable enzyme that catalyzes the amplification of a nucleic acid. Why should this render Taq unpatentable?
The Constitution does not require a claimed compound to have a formally “new” chemical structure or new function to justify a patent. Article I, section 8 of the Constitution authorizes patents “[t]o promote the Progress of Science and useful Arts . . . .” As explained by the Court:
Congress may not authorize the issuance of patents whose effects are to remove existent knowledge from the public domain, or to restrict free access to materials already available. Innovation, advancement, and things which add to the sum of useful knowledge are inherent requisites in a patent system which by constitutional command must ‘promote the Progress of useful Arts.’ This is the standard expressed in the Constitution and it may not be ignored.
Thus, the Constitution only limits patents that “remove existent knowledge from the public domain” or “restrict free access to materials already available.” Assuming that Taq was not previously known, a claim to it in isolated form simply cannot “remove existent knowledge from the public domain.” Because Taq naturally exists only in the context of a living organism, claiming it in “isolated” form cannot “restrict free access to” its source. Thus, constitutional limits cannot justify a prohibition on patents covering isolated naturally occurring products.
Nor does 35 U.S.C. § 101 clearly prohibit such patents. The statute specifically encompasses “discoveries,” so long as those discoveries relate to processes, compositions of matter, or articles of manufacture that are “new” and “useful.” In most cases, naturally occurring products are found in very minute quantities in complex association with other molecules inside living organisms. The act of isolating the natural product removes them from this context, thereby inevitably resulting in a composition that is materially different than anything that exists in nature. An “isolated” natural product therefore is “new” compared to the same product in its natural state. Its discovery thus could justify a claim under 35 U.S.C. § 101.
Finally, Supreme Court precedent does not clearly prohibit patenting of such claims. Under the closest Supreme Court precedent, a patent that is limited to a “non-naturally occurring article of manufacture or composition of matter” satisfies 35 U.S.C. § 101. Although it is often convenient to describe naturally occurring compounds in terms of chemical structure or nucleotide or amino acid sequence, they rarely if ever exist in nature as isolated compositions. Rather, they are found in complex associations with other compositions, usually within living organisms. The removal of these products from their natural context sometimes results in distinct chemical entities, such as the isolated nucleic acids in Myriad. Other times, the result is a highly purified form of the compound, such as isolated adrenaline or purified vitamin B12. In each case, however, the intervention of man is required to produce the “isolated” composition. Claims directed to “isolated” natural compounds thus are limited to purely artificial, non-naturally occurring compositions of matter. This should make them patentable, irrespective of whether they have a novel chemical structure or new utility in isolated form.
It is our sincere hope that the Court will not only find isolated nucleic acids to be patentable, but that it will do so under a rationale which allows for other naturally derived products to similarly be patentable. In as much as a possible test can be garnered, our recommendation is to find that a naturally derived product satisfies 35 U.S.C. § 101 as long as it is claimed in a purely man-made form (and thus is “new”), and the form in which it is claimed has a practical utility disclosed in the Specification (and thus is “useful”). This test closely aligns with the plain language of 35 U.S.C. § 101. Challenges to the eligibility of such claims could then focus on two clear issues: (1) whether the claim encompasses the product in its natural state; and (2) whether the claim is reasonably commensurate in scope with the disclosed utility (i.e., is the claim narrowly tailored to products that possess the disclosed utility?). This allows overly broad claims to be invalidated without resorting to a categorical ban on a broad class of subject matter. Moreover, it would not require courts to answer the philosophical question of whether something has enough of a structural or functional change to justify a patent.
NEW YORK (GenomeWeb News) – A federal appeals court today has for a second time reversed a lower district court’s decision that isolated genes are not patentable, but it also partly affirmed the District Court’s decision that certain methods patents “comparing” or “analyzing” gene sequences may not be patentable.
The Supreme Court recently asked the US Court of Appeals for the Federal Circuit to reconsider its earlier decision in the case, The Association for Molecular Pathology v. the US Patent and Trademark Office and Myriad Genetics, in light of its ruling in another lawsuit, called Mayo Collaborative Services v. Prometheus Laboratories.
AMP v USPTO focuses on the patentability of Myriad Genetics’ claims on isolated gene sequences and diagnostic methods related to its BRACAnalysis test. In Mayo v Prometheus, the Supreme Court recently invalidated patents held by diagnostics firm Prometheus because they merely described laws of nature, and did not apply those laws of nature in a markedly different manner as to warrant a patent.
Despite the Supreme Court’s ruling in Mayo, the CAFC in a 2-1 decision maintained that although isolated gene sequences may be derived from naturally occurring substances, their isolation requires human intervention in order to make them useful in medical care and so are deserving of patent protection.
“We are very pleased with the favorable decision the Court rendered today which again confirmed that isolated DNA is patentable,” Myriad Genetics President and CEO Peter Meldrum said in a statement. “Importantly, the court agreed with Myriad that isolated DNA is a new chemical matter with important utilities which can only exist as the product of human ingenuity.”
The decision was met with disappointment by those opposing gene patenting.
“It is extremely disappointing that despite the Supreme Court’s ruling, the appeals court has failed to fully re-consider the facts of this case,” Chris Hansen, a staff attorney with the ACLU Speech, Privacy and Technology Project, said in a statement.
The case against Myriad was filed in 2009 by the Public Patent Foundation, American Civil Liberties Union, AMP, and others who claim that patents cannot cover natural phenomena and that Myriad’s patents, and others like them, will hinder genetics research and keep some people from accessing tests and second opinions.
“This ruling prevents doctors and scientists from exchanging their ideas and research freely,” Hansen added the ACLU statement today. “Human DNA is a natural entity like air or water. It does not belong to any one company.”
Myriad said again today what it has argued all along, that gene patents have not thwarted research, that the cost of its BRACAnalysis test is not prohibitive and is covered through most insurance for “appropriate” patients, and that second opinion testing is available in many US labs.
“Certainly, you could hear a collective sigh of relief from the biotech industry, as of this decision,” Jennifer Camacho, an attorney and shareholder with law firm Greenberg Traurig, told GenomeWeb Daily News today.
“Isolated DNA patents remain intact. We still have patent eligibility for isolated DNA,” Camacho said, explaining that the court’s decision to uphold the patentability of isolated DNA may be seen by the biotech industry as more important than its reading of the reach of the Prometheus decision.
“They did actually take [the Prometheus decision] into consideration,” Camacho said, adding that it did not change the judges’ analysis.
“This puts a narrow interpretation of Prometheus in the books, as being limited to the ‘laws of nature’ exclusion, she added.
Camacho told GWDN that she was struck by how similar today’s CAFC ruling was to the original. She pointed out that part of one judge’s opinion, which argued that whether some patents should or should not be awarded are policy questions that are best left to Congress, was the same language as in the first opinion.
For Myriad, the ruling provided mixed results, Goldman Sachs Investment Research analyst Isaac Ro said in a note today.
On the positive side for Myriad, the patent eligibility of its BRCA1 and BRCA2-based tests was upheld again based on its isolated DNA claims and screening method claims. But a potential negative is that the CAFC also upheld the District Court’s opinion that Myriad’s method claims for comparing DNA sequences are not eligible.
“The outcome is modestly disappointing,” Ro stated, adding that the critical question now is whether or not the Supreme Court will agree to hear the case next year.
US Supreme Court Agrees to Hear Myriad Patent Case Again
NEW YORK (GenomeWeb News) – The US Supreme Court decided on Friday to once again hear the American Civil Liberty Union’s case against Myriad Genetics challenging the firm’s patent rights related to BRCA1 and BRCA2 genes.
The decision by the court to hear the case — originally filed by ACLU, the Public Patent Foundation, the Association for Molecular Pathology and others in 2009 — comes a little more than three months after a federal appeals courtissued a mixed ruling in which it found that isolated genes are patentable, but that certain methods patents that compare or analyze gene sequences may not be.
The US Court of Appeals for the Federal Circuit issued its decision in August after the Supreme Court asked it in March to reconsider a decision rendered by the appeals court in 2011 in light of the Supreme Court’s decision in another case, Mayo Collaborative Services v. Prometheus Laboratories. In that case, the Supreme Courtinvalidated patents held by Prometheus, saying the patents merely described laws of nature but did not apply those laws of nature in a markedly different manner as to warrant a patent.
The appeals court originally ruled in July 2011 that Myriad’s patents covering isolated DNA are patentable under Section 101 of the US Patent Act, reversing a decision by the Federal District Court for the Southern District of New York that isolated DNA is not much different from gene sequences found in nature and therefore is not patentable.
This past September, ACLU and the Public Patent Foundation asked the Supreme Court to once again take up the issue of whether Myriad’s claims on genes that predict the risk of ovarian and breast cancer can be patented. ACLU and the foundation contend that Myriad’s BRCA1 and BRCA2 gene patents should be invalidated because the genes are products of nature and allowing Myriad patent protection stifles scientific research and patient access to medical care.
“Myriad did not invent human genes, and has no right to claim ownership of them just because they removed them from the body,” Daniel Ravicher, executive director of PUBPAT, said in a statement on Friday. “The government does not have the right to give a corporation the exclusive power to control what we know about our own genetic makeup.”
Myriad President and CEO Peter Meldrum said in a statement, however, that patent protection is necessary to drive technological innovation.
“Two previous decisions by the Federal Circuit Court of Appeals confirmed the patentability of our groundbreaking diagnostic test that has helped close to 1 million people learn about their hereditary cancer risk,” he said. “Myriad devoted more than 17 years and $500 million to develop its BRACAnalysis test. The discovery and development of pioneering diagnostics and therapeutics require a huge investment and our US patent system is the engine that drives this innovation.
“This case has great importance for the hundreds of millions of patients whose lives are saved and enhanced by the life science industry’s products,” he said.
A typical cancer cell has thousands of mutations scattered throughout its genome and hundreds of mutated genes. However, only a handful of those genes, known as drivers, are responsible for cancerous traits such as uncontrolled growth. Cancer biologists have largely ignored the other mutations, believing they had little or no impact on cancer progression.
But a new study from MIT, Harvard University, the Broad Institute and Brigham and Women’s Hospital reveals, for the first time, that these so-called passenger mutations are not just along for the ride. When enough of them accumulate, they can slow or even halt tumor growth.
The findings, reported in this week’sProceedings of the National Academy of Sciences, suggest that cancer should be viewed as an evolutionary process whose course is determined by a delicate balance between driver-propelled growth and the gradual buildup of passenger mutations that are damaging to cancer, says Leonid Mirny, an associate professor of physics and health sciences and technology at MIT and senior author of the paper.
Furthermore, drugs that tip the balance in favor of the passenger mutations could offer a new way to treat cancer, the researchers say, beating it with its own weapon — mutations. Although the influence of a single passenger mutation is minuscule, “collectively they can have a profound effect,” Mirny says. “If a drug can make them a little bit more deleterious, it’s still a tiny effect for each passenger, but collectively this can build up.”
Lead author of the paper is Christopher McFarland, a graduate student at Harvard. Other authors are Kirill Korolev, a Pappalardo postdoctoral fellow at MIT, Gregory Kryukov, a senior computational biologist at the Broad Institute, and Shamil Sunyaev, an associate professor at Brigham and Women’s.
Power struggle
Cancer can take years or even decades to develop, as cells gradually accumulate the necessary driver mutations. Those mutations usually stimulate oncogenes such as Ras, which promotes cell growth, or turn off tumor-suppressing genes such as p53, which normally restrains growth.
Passenger mutations that arise randomly alongside drivers were believed to be fairly benign: In natural populations, selection weeds out deleterious mutations. However, Mirny and his colleagues suspected that the evolutionary process in cancer can proceed differently, allowing mutations with only a slightly harmful effect to accumulate.
To test this theory, the researchers created a computer model that simulates cancer growth as an evolutionary process during which a cell acquires random mutations. These simulations followed millions of cells: every cell division, mutation and cell death.
They found that during the long periods between acquisition of driver mutations, many passenger mutations arose. When one of the cancerous cells gains a new driver mutation, that cell and its progeny take over the entire population, bringing along all of the original cell’s baggage of passenger mutations. “Those mutations otherwise would never spread in the population,” Mirny says. “They essentially hitchhike on the driver.”
This process repeats five to 10 times during cancer development; each time, a new wave of damaging passengers is accumulated. If enough deleterious passengers are present, their cumulative effects can slow tumor growth, the simulations found. Tumors may become dormant, or even regress, but growth can start up again if new driver mutations are acquired. This matches the cancer growth patterns often seen in human patients.
“Cancer may not be a sequence of inevitable accumulation of driver events, but may be actually a delicate balance between drivers and passengers,” Mirny says. “Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations.”
When they analyzed passenger mutations found in genomic data taken from cancer patients, the researchers found the same pattern predicted by their model — accumulation of large quantities of slightly deleterious mutations.
Tipping the balance
In computer simulations, the researchers tested the possibility of treating tumors by boosting the impact of deleterious mutations. In their original simulation, each deleterious passenger mutation reduced the cell’s fitness by about 0.1 percent. When that was increased to 0.3 percent, tumors shrank under the load of their own mutations.
The same effect could be achieved in real tumors with drugs that interfere with proteins known as chaperones, Mirny suggests. After proteins are synthesized, they need to be folded into the correct shape, and chaperones help with that process. In cancerous cells, chaperones help proteins fold into the correct shape even when they are mutated, helping to suppress the effects of deleterious mutations.
Several potential drugs that inhibit chaperone proteins are now in clinical trials to treat cancer, although researchers had believed that they acted by suppressing the effects of driver mutations, not by enhancing the effects of passengers.
In current studies, the researchers are comparing cancer cell lines that have identical driver mutations but a different load of passenger mutations, to see which grow faster. They are also injecting the cancer cell lines into mice to see which are likeliest to metastasize.
Researchers in the Department of Genetics at the Harvard University Medical School have been awarded $500,000 by the National Institutes of Health for the first year of a four-year project to study multi-allelic copy number variation in the human genome.
As part of the research, the Harvard team is using a Bio-Rad QX100 Droplet Digital PCR system as one of two methods to analyze multi-allelic CNVs in human cohorts. The researchers are also using a computational method that compares available whole-genome sequencing data.
Steven McCarroll, a professor of genetics at Harvard Med and director of genetics at the Stanley Center for Psychiatric Research at the Broad Institute, is principal investigator on the grant, which is being administered by NIH’s National Human Genome Research Institute.
According to a recently published grant abstract, McCarroll and colleagues seek to analyze multi-allelic CNVs, which involve genes and other functional elements for which three or more segregating alleles give rise to a wide range of copy numbers — between two and 10 — per diploid human genome.
These multi-allelic CNVs have been “refractory to widely used analysis methods and are not assessed in the genome-scale molecular or statistical approaches used to study genetically complex phenotypes in humans,” the researchers wrote.
The project builds on research that McCarroll’s group previously conducted on characterizing multi-allelic duplication CNVs of a megabase-long inversion polymorphism in a particular locus of chromosome 17 called 17q21.31, which contains markers previously associated with female fertility, female meiotic recombination, and neurological disease.
As part of that research, published in the August 2012 issue of Nature Genetics, the group analyzed read depth in the locus by applying an algorithm called Genome Structure in Populations, or Genome STRiP, to whole-genome sequencing data from 946 unrelated individuals sampled as part of the 1000 Genomes Project; and used droplet-based digital PCR to analyze 120 parent-offspring trios from HapMap.
They found that their measurements of integer copy number varied from two to eight, and were 99.1 percent concordant across 234 genotypes in overlapping samples, thus validating both the computational and digital PCR methods.
More specifically, for the digital PCR assay, the group designed a pair of PCR primers and a dual-labeled fluorescence-FRET oligonucleotide probe to both the CNV locus and a two-copy control locus. Then they used a droplet generator from QuantaLife to compartmentalize the PCR reaction into uniform 1-nanoliter emulsion-based droplets containing zero, one, or very few template molecules for each locus; and a droplet reader from QuantaLife to count the number of positive and negative droplets, comparing the droplet counts of the CNV locus to the control locus to determine absolute copy number.
QuantaLife originally developed the droplet-based digital PCR system, but was acquired in October by Bio-Rad, which rebranded the platform as the QX100 Droplet Digital PCR system (PCR Insider, 10/6/2011).
Annette Tumolo, director of the digital biology center at Bio-Rad, told PCR Insider this week that McCarroll has access to two such platforms, one of which is in use at Harvard and was obtained from QuantaLife, and one of which Bio-Rad sold to the Broad Institute.
Tumolo said that Bio-Rad maintains “an active and positive relationship” with the McCarroll lab. “They’ve gotten great results [with the QX100], and were able to rapidly publish the Nature Genetics paper,” Tumolo said.
Under the new NHGRI grant, McCarroll and colleagues plan to “accurately analyze mCNVs in reference populations” using both the computational and digital PCR approach, the researchers wrote in their grant abstract.
“By analyzing these data in a statistical framework that incorporates information about genotypes, allele frequencies, inheritance, and haplotypes, we will place mCNV alleles onto the haplotype maps created by HapMap and 1000 Genomes, and render mCNVs accessible to genotype imputation to the fullest extent possible,” the grant abstract states.
In addition, McCarroll’s group hopes to “deeply characterize mCNVs at 10 biomedically important loci, to understand these polymorphisms at the levels of population genetics, mutational rates and histories, and relationships to clinical phenotypes. Finally, we will pilot inexpensive in silico genome-wide association studies for mCNVs based on statistical imputation into existing GWAS data sets.”
The end goal of the project is to discover relationships between disease risk and gene dosage, which will help reveal the molecular etiology of human disease, the researchers wrote.
Ben Butkus is senior editor of GenomeWeb’s premium content and the editor of PCR Insider. He covers technologies and trends in PCR, qPCR, nucleic acid amplification, and sample prep. E-mail him here or follow his GenomeWeb Twitter account at@PCRInsider.
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