Posts Tagged ‘Hematopoietic stem cells’

Targeting hematopoietic stem cells

Larry H. Bernstein, MD, FCAP, Curator



New technology uncovered the stem cell niche in the bone marrow

HSCs, Stem cells, hematopoiesis

Hematopoietic stem cells (HSCs) are so rare that it’s difficult to comprehensively localize dividing and non-dividing HSCs. Thus, there has controversy about their specific location in the bone marrow. A recent Nature publication reported that the HSCs resides mainly in perisinusoidal niches through out the bone marrow and there are no spatially distinct niches for dividing and non-dividing blood-forming stem cells. This group of researchers at UT Southwestern Medical Center started the generation of a GFP knock-in for the gene Ctnnal1, a generic marker for HSCs in mice (α-catulinGFP mice) and confirmed that α-catulin-GFP+c-kit+ cells represent blood-forming HSCs by showing that α-catulin-GFP+c-kit+ cells gave long term multi-lineage reconstitution of irradiated mice. Using a tissue-clearing technique and deep confocal imaging, they were able to image thousands of α-catulin-GFP+c-kit+ cells and see their relation to other cells. This publication improved the understanding of the microenvironment of HSCs in the bone marrow, which would significantly improve the safety and effectiveness of bone marrow transplantation.

Melih Acar, etc. (October 2015) Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature


Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal

AcarKS. KocherlakotaMM. MurphyJG. PeyerH OguroCN. InraC JaiyeolaZ ZhaoK Luby-Phelps & Sean J. Morrison
Nature526,126–130(01 October 2015)


Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial1. HSCs are rare and few can be found in thin tissue sections2, 3 or upon live imaging4, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as αcatulinGFP), we discover that αcatulinGFP is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of αcatulin−GFP+c-kit+ cells give long-term multilineage reconstitution of irradiated mice, indicating thatαcatulin−GFP+c-kit+ cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of αcatulin–GFP+c-kit+ cells and to digitally reconstruct large segments of bone marrow. The distribution of αcatulin–GFP+c-kit+ cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr+) and Cxcl12high niche cells, and approximately 85% of HSCs were within 10 μm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67+) and non-dividing (Ki-67), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr+Cxcl12high cells throughout the bone marrow.


Figure 1: Deep imaging of αcatulin−GFP+ HSCs in digitally reconstructed bone marrow.close


Deep imaging of [agr]-catulin-GFP+ HSCs in digitally reconstructed bone marrow.

a, Only 0.021 ± 0.006% of αcatulinGFP/+ bone marrow cells were GFP+ (n = 14 mice in 11 independent experiments). b, Nearly allαcatulin−GFP+c-kit+ bone marrow cells were CD150+CD48 (n = 9 mice in 3 independent experiments;


Extended Data Figure 3: αcatulin−GFP expression among haematopoietic cells is highly restricted to HSCs.


[agr]-catulin-GFP expression among haematopoietic cells is highly restricted to HSCs.


a, The frequency of αcatulin−GFP+ bone marrow cells in negative control αcatulin+/+ (WT) mice and α-catulinGFP/+ mice (n = 14 mice per genotype in 11 independent experiments). In all cases in this figure, percentages refer to the frequency of each population as a percentage of WBM cells. b, αcatulin−GFP+c-kit+ cells from Fig. 1b are shown (blue dots) along with all other bone marrow cells in the same sample (red dots). c, CD150+CD48LSK HSCs express αcatulin−GFP but CD150CD48LSK MPPs do not (n = 17 mice in 12 independent experiments). A minority of the αcatulin−GFP+c-kit+ cells had high forward scatter, lacked reconstituting potential, and were gated out when isolating HSCs by flow cytometry and when identifying HSCs during imaging (see Extended Data Fig. 5for further explanation). d, Linc-kitlowSca-1lowCD127+CD135+ common lymphoid progenitors (CLPs), Linc-kit+Sca-1CD34+CD16/32 common myeloid progenitors (CMPs), Linc-kit+Sca-1CD34+CD16/32+ granulocyte-macrophage progenitors (GMPs), and Linc-kit+Sca-1CD34CD16/32 megakaryocyte-erythroid progenitors (MEPs) did not express αcatulin−GFP. αcatulinGFP/+ and control cell populations had similar levels of background GFP signals that accounted for fewer than 1% of the cells in each population (n = 9 mice per genotype in 2 independent experiments).


Extended Data Figure 7: HSC density is higher in the diaphysis as compared to the metaphysis.

HSC density is higher in the diaphysis as compared to the metaphysis.

a, Schematic of a femur showing the separation of epiphysis/metaphysis from diaphysis. We divided metaphysis from diaphysis at the point where the central sinus branched (see red line in panels a, f,and i). This is also the point at wh…



Extended Data Figure 9: Bone marrow blood vessel types can be distinguished based on vessel diameter, continuity of basal lamina, morphology, and position; and no difference in the distribution of HSCs in the bone marrow of male and female mice was detected.close

Bone marrow blood vessel types can be distinguished based on vessel diameter, continuity of basal lamina, morphology, and position; and no difference in the distribution of HSCs in the bone marrow of male and female mice was detected.

a, b, Schematic (a) and properties (b) of blood vessels in the bone marrow. Blood enters the marrow through arterioles that branch as they become smaller in diameter and approach the endosteum, where they connect to smaller diameter tra…


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Larry H. Bernstein, MD, FCAP, Curator



Hematopoietic Stem Cells Use a Simple Heirarchy




These papers challenge this model by arguing that the CMP does not exist. Let me say that again – the CMP, a cell that has been identified several times in mouse and human bone marrow isolates, does not exist. When CMPs were identified from mouse and human none marrow extracts, they were isolated by means of flow cytometry, which is a very powerful technique, but relies on the assumption that the cell type you want to isolate is represented by the cell surface protein you have chosen to use for its isolation. Once the presumptive CMP was isolated, it could recapitulate the myeloid lineage when implanted into the bone marrow of laboratory animals and it could also produce all the myeloid cells in cell culture. Sounds convincing doesn’t it?

In a paper in Science magazine, Faiyaz Notta and colleagues from the University of Toronto beg to differ. By using a battery of antibodies to particular cell surface molecules, Notta and others identified 11 different cell types from umbilical cord blood, bone marrow, and human fetal liver that isolates that would have traditionally been called the CMP. It turns out that the original CMP isolate was a highly heterogeneous mixture of different cell types that were all descended from the HSC, but had different developmental potencies.


Notta and others used single-cell culture assays to determine what kinds of cells these different cell types would make. Almost 3000 single-cell cultures later, it was clear that the majority of the cultured cells were unipotent (could differentiate into only one cell type) rather than multipotent. In fact, the cell that makes platelets, the megakarocyte, seems to derive directly from the MPP, which jives with the identification of megakarocyte progenitors within the HSC compartment of bone marrow that make platelets “speedy quick” in response to stress (see R. Yamamoto et al., Cell 154, 1112 (2013); S. Haas, Cell Stem Cell 17, 422 (2015)).

Another paper in the journal Cell by Paul and others from the Weizmann Institute of Science, Rehovot, Israel examined over 2700 mouse CMPs and subjected these cells to gene expression analyses (so-called single-cell transriptome analysis). If the CMP is truly multipotent, then you would expect it to express genes associated with lots of different lineages, but that is not what Paul and others found. Instead, their examination of 3461 genes revealed 19 different progenitor subpopulations, and each of these was primed toward one of the seven myeloid cell fates. Once again, the presumptive CMPs looked very unipotent at the level of gene expression.

One particular subpopulation of cells had all the trappings of becoming a red blood cell and there was no indication that these cells expressed any of the megakarocyte-specific genes you would expect to find if MEPS truly existed. Once again, it looks as though unipotency is the main rule once the MPP commits to a particular cell lineage.

Thus, it looks as though either the CMP is a very short-lived state or that it does not exist in mouse and human bone marrow. Paul and others did show that cells that could differentiate into more than one cell type can appear when regulation is perturbed, which suggests that under pathological conditions, this system has a degree of plasticity that allows the body to compensate for losses of particular cell lineages.

 A model of the changes in human My-Er-Mk differentiation that occur across developmental time points. Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.
A model of the changes in human My-Er-Mk differentiation that occur across developmental time points.
Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.

Fetal HSCs, however, are a bird of a different feather, since they divide quickly and reside in fetal liver.  Also, these HSCs seem to produce CMPs, which is more in line with the classical model.  Does the environmental difference or fetal liver and bone marrow make the difference?  In adult bone marrow, some HSCs nestle next to blood vessels where they encounter cells that hang around blood vessels known as “pericytes.”  These pericytes sport a host of cell surface molecules that affect the proliferative status of HSCs (e.g., nestin, NG2).  What about fetal liver?  That’s not so clear – until now.

In the same issue of Science magazine, Khan and others from the Albert Einstein College of Medicine in the Bronx, New York, report that fetal liver also has pericytes that express the same cell surface molecules as the ones in bone marrow, and the removal of these cells reduces the numbers of and proliferative status of fetal liver HSCs.

Now we have a conundrum, because the same cells in bone marrow do not drive HSC proliferation, but instead drive HSC quiescence.  What gives? Khan and others showed that the fetal liver pericytes are part of an expanding and constantly remodeling blood system in the liver and this growing, dynamic environment fosters a proliferative behavior in the fetal HSCs.

When umbilical inlet is closed at birth, the liver pericytes stop expressing Nestin and NG2, which drives the HSCs from the fetal liver to the other place were such molecules are found in abundance – the bone marrow.

These models give us a better view of the inner workings of HSC differentiation.  Since HSC transplantation is one of the mainstays of leukemia and lymphoma treatment, understanding HSC biology more perfectly will certainly yield clinical pay dirt in the future.


Distinct routes of lineage development reshape the human blood hierarchy across ontogeny

In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34+ cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead, only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult “two-tier” hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.

Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors

Franziska Paul, et al.
Cell Dec 2015; Volume 163, Issue 7:1663–1677
Figure thumbnail fx1
  • Transcriptionally primed single-cell subpopulations in early myeloid progenitors
  • Transcription factors and epigenetic landscapes that regulate myeloid priming
  • Mixed lineage states are not observed but appear when regulation is perturbed
  • New reference model for studying hematopoiesis at single-cell resolution



Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.



Fetal liver hematopoietic stem cell niches associate with portal vessels

Jalal A. Khan, et al.   Science  08 Jan 2016; 351(6269):176-180
How HSCs populate the fetal liver

Hematopoietic stem cells (HSCs) undergo dramatic expansion in the fetal liver before migrating to their definitive site in the bone marrow. Khan et al. identify portal vessel–associated Nestin+NG2+ pericytes as critical HSC niche components (see the Perspective by Cabezas-Wallscheid and Trumpp). The portal vessel niche and HSCs expand according to fractal geometries, suggesting that niche cells—rather than factors expressed by the niche—drive HSC proliferation. After birth, arterial portal vessels transform into portal veins, and lose Nestin+NG2+pericytes. When this happens, the niche is lost and HSCs migrate away from the neonatal liver.

Science, this issue p. 176; see also p. 126


Whereas the cellular basis of the hematopoietic stem cell (HSC) niche in the bone marrow has been characterized, the nature of the fetal liver niche is not yet elucidated. We show that Nestin+NG2+ pericytes associate with portal vessels, forming a niche promoting HSC expansion. Nestin+NG2+ cells and HSCs scale during development with the fractal branching patterns of portal vessels, tributaries of the umbilical vein. After closure of the umbilical inlet at birth, portal vessels undergo a transition from Neuropilin-1+Ephrin-B2+ artery to EphB4+ vein phenotype, associated with a loss of periportal Nestin+NG2+ cells and emigration of HSCs away from portal vessels. These data support a model in which HSCs are titrated against a periportal vascular niche with a fractal-like organization enabled by placental circulation.



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Understanding the Stem Cell Niche: A Webinar by The Scientist

Reporter: Stephen J. Williams, Ph.D.


The Scientist

nature stem cell

Schematic diagram showing some of the factors implicated in each process. Haematopoietic stem cells (HSCs) bound to the bone-marrow niche are mobilized in response to granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide, or after peripheral myeloablation following treatment with 5-fluorouracil (5-FU). After extravasation from the bone-marrow cords into the microvasculature, HSCs enter the circulation and are distributed to peripheral tissues such as the spleen or liver. HSCs locate close to endothelial cells in the splenic red pulp. They home to the bone-marrow cords through the circulation, a process that is controlled by a number of adhesion molecules such as very late antigen 4 (VLA4), VLA5, lymphocyte function-associated antigen 1 (LFA1) or selectins. After entering the bone marrow, HSCs specifically lodge in the niche, a process requiring membrane-bound stem-cell factor (SCF), CXC-chemokine ligand 12 (CXCL12), osteopontin (OPN), hyaluronic acid, and their corresponding receptors. CXCR4, CXC-chemokine receptor 4; E-selectin, endothelial-cell selectin; P-selectin, platelet selectin; PSGL1, P-selectin glycoprotein ligand 1.


Understanding the Stem Cell Niche

  This presentation will begin on Tuesday, December 01, 2015 at 02:30 PM Eastern Standard Time.

Free Webinar
Tuesday December 1, 2015
2:30 – 4:00 PM EST

Stem cells provide an attractive model to study human physiology and disease. However, technical challenges persist in the biological characterization and manipulation of stem cells in their native microenvironment. The Scientist brings together a panel of experts to discuss interactions between stem cells and external cues, and the role of the stem cell niche in development and disease. Topics to be covered include the molecular mechanisms of hematopoietic stem cell niche interactions and techniques for engineering 3-D stem-cell microenvironments. Following the presentations, attendees will have an opportunity to ask questions concerning their specific applications and receive answers in real-time.


Dr. Jon Hoggatt, Assistant Professor of Medicine, Cancer Center and Center for Transplantation Sciences, Harvard Medical School/Massachusetts General Hospital.

Dr. Todd McDevitt, Senior Investigator, Gladstone Institute of Cardiovascular Disease, Professor, Department of Bioengineering & Therapeutic Sciences, UCSF.


Understanding the Stem Cell Niche
Click Here To Watch The Video

To find out about our upcoming events follow us on Twitter @LabMgrEvents


Notes from Webinar:

Hematopoetic stem cells good model since now we have liquid biopsies (as a result field has skyrocketed).

Two processes involved with stem cells finding their niche

  1. Homing; CXCR4-SDK1 dependent process into the bone marrow.
  2. Mobilization: stem cells moving from bone into blood (found that GMCSF main factor responsible for this process)

Dr. Raymond Schofield was one of the first to propose the existence of this stem cell niche (each progenitor will produce a unique factor {possibly a therapeutic target} for example leptin+ receptor target perivascular cells so one target is good for only a small subset of stem cells)

Therefore it may be possible or advantageous to target the whole stem cell milieu. One such possible target they are investigating is CD26 (dipeptyl peptidase). The diabetes drug Januvia is an inhibitor of CD26.

It was also noticed if inhibit the GMCSF receptor complex can inhibit the whole stem cell niche.

Prostoglandins and stem cell niche

  • Indomethacin blocks the mobilization step
  • Prostaglandin E increases homing
  • GMCSF and malaxocam (COX2 inhibitor) flattens osteoblast cells and may be a mechanism how inhibition of prostaglandin synthesis blocks mobilization
  • Found that the PGE4 receptor is ultimately responsible for the NSAID effect

The niche after G-CSF

Dr. Hoggat found that macrophages are supplying the factors that support the niche. He will be presenting the findings at 2015 Hematology conference. (See information about his conference presentation here).

From the 57th Annual American Society of Hematology Meeting (2015) please see Dr. Hoggat’s moderated section Hematopoiesis and Stem Cells: Microenvironment, Cell Adhesion and Stromal Stem Cells: Hematopoietic Stem Cell Niche


Relevant articles from Dr. Hoggat

Anti-CD47 Therapy Is More Than a Dinner Bell October 19, 2015

Dr. Hoggatt looks at the therapeutic effects of blocking CD47 aside from alerting macrophages to devour tumor cells.

Hematopoietic Stem Cells Should Hold Their Breath August 12, 2015

Dr. Hoggatt and Hannah Rasmussen discuss new approaches to the use of hematopoietic stem cells considering observer effects that emerge due to our experimental systems for HSCs.

Prostaglandin E2 enhances hematopoietic stem cell homing, survival, and proliferation. Hoggatt J, Singh P, Sampath J, Pelus LM. Blood. 2009 May 28;113(22):5444-55. doi: 10.1182/blood-2009-01-201335. Epub 2009 Mar 26.


Prostaglandin E2 enhances long-term repopulation but does not permanently alter inherent stem cell competitiveness. Hoggatt J, Mohammad KS, Singh P, Pelus LM. Blood. 2013 Oct 24;122(17):2997-3000. doi: 10.1182/blood-2013-07-515288. Epub 2013 Sep 18.


Pharmacologic increase in HIF1α enhances hematopoietic stem and progenitor homing and engraftment. Speth JM, Hoggatt J, Singh P, Pelus LM. Blood. 2014 Jan 9;123(2):203-7. doi: 10.1182/blood-2013-07-516336. Epub 2013 Oct 28.


Blockade of prostaglandin E2 signaling through EP1 and EP3 receptors attenuates Flt3L-dependent dendritic cell development from hematopoietic progenitor cells. Singh P, Hoggatt J, Hu P, Speth JM, Fukuda S, Breyer RM, Pelus LM. Blood. 2012 Feb 16;119(7):1671-82. doi: 10.1182/blood-2011-03-342428. Epub 2011 Nov 22.


Recovery from hematopoietic injury by modulating prostaglandin E(2) signaling post-irradiation. Hoggatt J, Singh P, Stilger KN, Plett PA, Sampson CH, Chua HL, Orschell CM, Pelus LM. Blood Cells Mol Dis. 2013 Mar;50(3):147-53. doi: 10.1016/j.bcmd.2012.11.006. Epub 2012 Nov 30.


Pulse exposure of haematopoietic grafts to prostaglandin E2 in vitro facilitates engraftment and recovery. Pelus LM, Hoggatt J, Singh P. Cell Prolif. 2011 Apr;44 Suppl 1:22-9. doi: 10.1111/j.1365-2184.2010.00726.x.


Pleiotropic effects of prostaglandin E2 in hematopoiesis; prostaglandin E2 and other eicosanoids regulate hematopoietic stem and progenitor cell function. Pelus LM, Hoggatt J. Prostaglandins Other Lipid Mediat. 2011 Nov;96(1-4):3-9. doi: 10.1016/j.prostaglandins.2011.06.004. Epub 2011 Jun 21. Review.


Differential stem- and progenitor-cell trafficking by prostaglandin E2. Hoggatt J, Mohammad KS, Singh P, Hoggatt AF, Chitteti BR, Speth JM, Hu P, Poteat BA, Stilger KN, Ferraro F, Silberstein L, Wong FK, Farag SS, Czader M, Milne GL, Breyer RM, Serezani CH, Scadden DT, Guise TA, Srour EF, Pelus LM. Nature. 2013 Mar 21;495(7441):365-9. doi: 10.1038/nature11929. Epub 2013 Mar 13.


Eicosanoid regulation of hematopoiesis and hematopoietic stem and progenitor trafficking.Hoggatt J, Pelus LM. Leukemia. 2010 Dec;24(12):1993-2002. doi: 10.1038/leu.2010.216. Epub 2010 Sep 30. Review.


Hematopoietic stem cell mobilization with agents other than G-CSF. Hoggatt J, Pelus LM. Methods Mol Biol. 2012;904:49-67. doi: 10.1007/978-1-61779-943-3_4.


Mobilization of hematopoietic stem cells from the bone marrow niche to the blood compartment. Hoggatt J, Pelus LM. Stem Cell Res Ther. 2011 Mar 14;2(2):13. doi: 10.1186/scrt54. Review.


Engineering 3D Pluripotent Stem Cell Microenvironments by Todd McDevitt, Ph.D.

In recent years, it has finally been shown how to produce centrally derived (self assembling) organoids (microtissues).


How to specifically deliver specific morphogens in 3D organoids


  1. Microparticle (MP)-mediated delivery (can do in mouse and human): reduces the amount needed to be delivered



What are other effects of introduced MP in ES (embryonic stem cell) aggregates?

  1. a) physiocomechanical changes –mechanical effects of materials
  2. b) how changes in local presentation of factors affect bioavailbility and binding properties







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Writer and curator: Larry H. Bernstein, MD, FCAP and
Curator: Aviva Lev-Ari, PhD, RN

There is an explosion of work-in-progress in applications to regenerative medicine using inducible pluripotent stem cells in both endothelial and cardiomyocyte postischemic repair, and also in post bone marrow radiation restoration, with benefits and hazards.  The following article is quite novel in that it deals with stem cell regulation by DNA methylation.  Therefore, it deals with the essentiality of methylation of DNA in epigenetic regulation.

This is the fourth discussion of a several part series leading from the genome, to protein synthesis (1), posttranslational modification of proteins (2), examples of protein effects on metabolism and signaling pathways (3), and leading to disruption of signaling pathways in disease (4), and effects leading to mutagenesis.

1.  A Primer on DNAand DNA Replication

2.  Overview of translational medicine

3.  Genes, proteomes, and their interaction

4. Regulation of somatic stem cell Function

5.  Proteomics – The Pathway to Understanding and Decision-making in Medicine

6.  Genomics, Proteomics and standards

7.  Long Non-coding RNAs Can Encode Proteins After All

8.  Proteins and cellular adaptation to stress

9.  Loss of normal growth regulation


Posttranslational modification is a step in protein biosynthesis. Proteins are created by ribosomes translating mRNA into polypeptide chains. These polypeptide chains undergo
PTM before becoming the mature protein product.

Regulation of somatic stem cell Function by DNA Methylation and Genomic Imprinting

Mo Li1, Na Young Kim1, Shigeo Masuda1 and Juan Carlos izpisua Belmonte1,2 1Salk institute for Biological Studies, 10010 N Torrey Pines Rd, La Jolla, CA 92037, USA. 2Center of Regenerative Medicine in Barcelona, Dr Aiguader, 88, 08003 Barcelona, Spain. Corresponding author email:

Cell & Tissue Transplantation & Therapy 2013:5 19–23
This article is available from


Epigenetic regulation is essential for self-renewal and differentiation of somatic stem cells, including

  • hematopoietic stem cells (HSCs) and
  • neural stem cells (NSCs).

The role of DNA methylation, a key epigenetic pathway,

  • in regulating somatic stem cell function
    • under physiological conditions and during aging

has been intensively investigated.

Accumulating evidence highlights the dynamic nature of

  • the DNAmethylome
    • during lineage commitment of somatic stem cells and
  • the pivotal role of DNAmethyltransferases in
    • stem cell self-renewal and differentiation.

Recent studies on genomic imprinting have shed light on

  • the imprinted gene network (IGN) in somatic stem cells,
  1. where a subset of imprinted genes remain expressed and
  2. are important for maintaining self-renewal of these cells.

Together with emerging technologies, elucidation of the epigenetic mechanisms regulating somatic stem cells with normal or pathological functions may contribute to the development of regenerative medicine.

Keywords: somatic stem cells, epigenetics, DNA methylation, genomic imprinting, hematopoietic stem cells, neural stem cells


In adult animals, somatic stem cells (also known as adult stem cells) are responsible for maintaining tissue homeostasis and participate in tissue regeneration under injury conditions. Self-renewal and differentiation are two important aspects of somatic stem cell function. Epigenetic mechanisms underlying these processes have been intensively investigated. With the increasing ability

  • to identify and manipulate somatic stem cell populations from diverse tissues,
  • it is possible to dissect the epigenetic pathways that are
  1. either unique for a specific tissue or
  2. universally important in regulating stemness and differentiation.

Epigenetic control of somatic stem cell function exists at various levels, including

  • DNA methylation,
  • histone modification, and
  • higher-order chromatin structure dynamics.

Here, we focus on recent progress in our understanding of how

  • DNA methylation regulates somatic stem cell function.

DNA Methylation and stem cell Function

The role of DNA methylation in somatic stem cell compartments has gained increasing attention. Recent  evidence has shown that

  • DNA methylation is dynamically regulated during somatic stem cell differentiation and aging.1

A study of methylomes of human hematopoietic stem cells (HSCs) and two mature hematopoietic lineages,

  • including B cells and neutrophils, showed that
    • hypomethylated regions of lineage-specific genes often become methylated in opposing lineages, and that
    • progenitors display an intermediate methylation pattern

that is poised for lineage-specific resolution.2

Another study compared genome-wide promoter DNA methylation in human cord blood hematopoietic progenitor cells (HPCs) with

  • that in mobilized peripheral blood HPCs from aged individuals.

It was found that aged HPCs lose DNA methylation in a subset of genes that are hypomethylated in differentiated myeloid cells and

  • gain de novo DNA methylation at polycomb repressive complex 2 (PRC2) target sites.3

It was hypothesized that such epigenetic changes contribute to age-related loss of HSC function, such as a bias toward myeloid lineages. Recently, Beerman et al. studied the global DNA methylation landscape of HSCs in the context of

  • age-associated decline of HSC function.4

Over- all, the DNA methylation landscape remains stable during HSC ontogeny. However, HSCs isolated from old mice display higher global DNA methylation. Interestingly, they observed

  • localized DNA methylation changes in genomic regions associated with hematopoietic lineage differentiation.

These methylation changes preferentially map to genes

  • that are expressed in downstream progenitor and effector cells.

For example, genes that are important for the lymphoid and erythroid lineages

  • become methylated in “old” HSCs,

which is consistent with

  • the decline of lymphopoiesis and erythropoiesis during aging.

Additionally, inducing HSC proliferation by 5-fluorouracil treatment or

  • by limiting the number of transplantedHSCs
    • recapitulates the functional decline and DNA methylation changes during physiological aging.

A closer examination of the overlapping genes with significant DNA methylation changes during aging or enforced proliferation showed

  • an enrichment of DNA hypermethylation at PRC2 target loci,

echoing the observation by Bocker et al. in human HSCs.

Interestingly, a recent report showed that epigenetic alterations such as DNA hypermethylation that are accrued during aging,

  • can be fully reset by somatic reprogramming,

raising an interesting possibility that these aging-related epigenetic defects may be reserved by small molecules.5

Methylation of cytosines at CpG dinucleotides is catalyzed by three key enzymes.

DNA (cytosine-5)- methyltransferase 1 (DNMT1) is responsible for maintaining DNA methylation patterns during DNA replication

  • by methylating the newly synthesized hemi-methylated DNA.

The other two DNA methyltransferases, DNMT3a and DNMT3b,

  • are not DNA replication-dependent and can methylate fully unmethylated DNA de novo.

They are responsible for establishing new DNA methylation patterns during development.

DNMT3a, a gene required for neurogenesis,

  • is expressed in postnatal neural stem cells (NSCs).

In NSCs, DNMT3a methylates non-proximal promoter regions, such as gene bodies and intergenic regions. Surprisingly, rather than silencing gene expression,

DNMT3a-mediated DNA methylation in gene bodies antagonizes Polycomb-dependent repression and

  • facilitates the expression of neurogenic genes.6

The role of DNMT3a in HSCs has also been investigated. Both Dnmt3a and Dnmt3b are expressed in HSCs. An earlier study did not identify any defects in HSC function when Dnmt3a or Dnmt3b was removed.  However,

  • HSCs lackingboth of these de novomethyltransferases
    • fail to self-renew, yet retain the capacity to differentiate.7

A more recent study re-examined

  • the consequences of Dnmt3a loss in HSCs and
  • uncovered a progressive defect in differentiation that is only manifested during serial transplantation.8

At the molecular level, while Dnmt3a loss results in the expected hypomethylation at some loci,

  • it counterintuitively causes hypermethylation in even more regions.8

This seemingly paradoxical result echoes the  unconventional role of Dnmt3a in transcriptional  activation in NSCs (as discussed above). Both cases suggest a more complex regulatory function of DNMT3a that is

  • beyond simply methylating DNA.

In contrast, the loss of Dnmt1 produces more dramatic and immediate phenotypes in HSCs, manifested

  • in premature HSC exhaustion and
  • block of lymphoid differentiation,

highlighting the distinct requirements for different DNA methyltransferases in HSCs.9,10

Genomic Imprinting and stemness

DNA methylation also underlies genomic imprinting, which is an

  • evolutionarily conserved epigenetic mechanism of ensuring appropriate gene dosage during development.

One allele of the imprinted genes is

  • epigenetically marked by DNA methylation to be silenced according to the parental origin.

The pattern of imprinting

  • is established in germ cells and maintained in somatic cells.

Imprinted genes are thought to play critical roles in organismal growth and are relatively downregulated after birth.11 Recently, a series of reports demonstrated that

  • a subset of imprinted genes belonging to the purported imprinted gene network (IGN)12
  • remain expressed in somatic stem cells and
  • are important for maintaining self-renewal of these cells.

Through gene expression profiling, one group identified that several members of the IGN are expressed in

  1. murine muscle,
  2. epidermal, and
  3. long-term hematopoietic stem cells
  4. as well as in human epidermal and hematopoietic stem cells.13

In particular, the paternally expressed gene 3 (Peg3) gene was shown by another group

  • to mark cycling and quiescent stem cells in a wide variety of mouse tissues.14

The role of imprinted genes in regulating somatic stem cell function has been examined in two types of tissues.

In bronchioalveolar stem cells (BASCs), a lung epithelial stem cell population,

  • expression of IGN members is required for their self-renewal.

Bmi1, a polycomb repressive  complex 1 (PRC1) subunit,

  • is essential for controlling the expression of imprinted genes in BASCs without affecting their imprinting status.15

In Bmi1 mutant BASCs,  many members of the IGN become derepressed,

  • including p57, H19, Dlk1, Peg3, Ndn, Mest, Gtl2, Grb10, Plagl1, and Igf2.

Knockdown of p57, which is the most differentially expressed imprinted gene between normal and mutant BASCs,

  • partially rescues the self-renewal defect of lung stem cells.

Interestingly, insufficient levels of p57 also inhibit self-renewal of lung stem cells. Because p57 expression

  • remains monoallelic in Bmi1 knockdown cells,
  • Bmi1 is thought to maintain an appropriate level of expression from the expressed allele of p57.15

Another IGN member- delta-like homologue 1 (Dlk1) has been shown to be important for postnatal neurogenesis. Interestingly, in this context,

  • Dlk1 loses its imprinting in postnatal neural stem cells and niche astrocytes.16

These studies suggest that modulating IGN may represent another

  • epigenetic mechanism for balancing self-renewal and differentiation in somatic stem cells.

Thus, somatic stem cells either co-opt or remodel these developmental pathways involving the IGN

  • to fulfill the needs of tissue homeostasis during the adult stage.

In summary, several factors participate in regulating the epigenome of somatic stem cells.

Perturbations in the epigenome of somatic stem cells,

  • either during organismal aging or under pathological conditions,

will tip the balance between self-renewal and differentiation of somatic stem cells (Fig. 1). A detailed understanding of the mechanisms underlying these changes will likely result in novel therapeutic approaches targeting somatic stem cells.

Figure 1. The epigenome of somatic stem cells is regulated by diverse factors.

Future perspectives The epigenetic mechanisms governing self-renewal and differentiation of somatic stem cells are likely to be complex because of the diverse needs of different tissues. It would be interesting to determine whether a common mechanism, such as the IGN, exists across different somatic stem cells. Additionally, study- ing epigenetic pathways that are specific to one type of somatic stem cell requires the isolation of these cells and their differentiated progeny, which is more practical in model organisms than in humans. Along these lines, developing robust in vitro culture methods for human somatic stem cells and protocols for differentiating these cells into specific lineages are critical for uncovering epigenetic pathways that are unique to human somatic stem cells. In recent years, the field has seen a great improvement in methods of directed differentiation of human embryonic stem cells and induced pluripotent stem cells (iPSCs). For example, it is relatively straightforward to produce high-purity cell populations that resemble neural stem cells or mesenchymal stem cells from iPSCs.17

These methodologies not only are useful for studying the normal function of somatic stem cells, but also provide an exciting opportunity for understanding the role of somatic stem cells in disease pathology and a platform to screen for drugs. A recent study under- scored the usefulness of this approach. Liu et al. studied neural stem cells derived from Parkinson’s disease human iPSCs and uncovered previously unknown defects in nuclear morphology and epigenetic regulation in these derived NSCs.18 The cellular defects only menifest in “aged” neural stem cells, which is consistent with the fact that Parkinson’s disease pri- marily manifests in old age. More  importantly, this study identified neural stem cell as a potential target of therapeutic intervention for Parkinson’s disease.

Targeted modification of the human genome is  another technological advancement that is on the horizon to greatly facilitate the dissection of epige- netic pathways in somatic stem cells. Although gene targeting in somatic stem cells has been historically challenging, there have been encouraging successful reports following development of new genome-e diting technologies, such as Helper-dependent adenovi- ral vectors, TALENs, and CAS9/CRISPR. With the development of these new technologies, it seems that the stage has been set for a new wave of discoveries in epigenetic mechanisms of somatic stem cells.


1. Li M, Liu GH, Izpisua Belmonte JC. Navigating the epigenetic landscape of pluripotent stem cells. Nat Rev Mol Cell Biol. 2012;13(8):524–535.

2. Hodges E, Molaro A, Dos Santos CO, et al. Directional DNA methylation changes and complex intermediate states accompany lineage specificity in the adult hematopoietic compartment. Mol Cell. 2011;44(1):17–28.

3. Bocker MT, Hellwig I, Breiling A, Eckstein V, Ho AD, Lyko F. Genome- wide promoter DNA methylation dynamics of human hematopoietic progen- itor cells during differentiation and aging. Blood. 2011;117(19):e182–e189.

4. Beerman I, Bock C, Garrison BS, et al. Proliferation-dependent alterations of the DNA methylation landscape underlie hematopoietic stem cell aging. Cell Stem Cell. 2013;12(4):413–425.

5. Wahlestedt M, Norddahl GL, Sten G, et al. An epigenetic component of hematopoietic stem cell aging amenable to reprogramming into a young state. Blood. 2013;121(21):4257–4264.

6. Wu H, Coskun V, Tao J, et al. Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes. Science. 2010; 329(5990):444–448.

7. Tadokoro Y, Ema H, Okano M, Li E, Nakauchi H. De novo DNA meth- yltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells. J Exp Med. 2007;204(4):715–722.

8. Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet. 2011;44(1):23–31.

9. Broske AM, Vockentanz L, Kharazi S, et al. DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction. Nat Genet. 2009;41(11):1207–1215.

10. Trowbridge JJ, Snow JW, Kim J, Orkin SH. DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells. Cell Stem Cell. 2009;5(4):442–449.

11. Wood AJ, Oakey RJ. Genomic imprinting in mammals: emerging themes and established theories. PLoS Genet. 2006;2(11):e147.

12. Lui JC, Finkielstain GP, Barnes KM, Baron J. An imprinted gene network that controls mammalian somatic growth is down-regulated during postna- tal growth deceleration in multiple organs. Am J Physiol Regul Integr Comp Physiol. 2008;295(1):R189–R196.

13. Berg JS, Lin KK, Sonnet C, et al. Imprinted genes that regulate early mam- malian growth are coexpressed in somatic stem cells. PLoS One. 2011; 6(10):e26410.

14. Besson V, Smeriglio P, Wegener A, et al. PW1 gene/paternally expressed gene 3 (PW1/Peg3) identifies multiple adult stem and progenitor cell popu- lations. Proc Natl Acad Sci U S A. 2011;108(28):11470–11475.

15. Zacharek SJ, Fillmore CM, Lau AN, et al. Lung stem cell self-renewal relies on BMI1-dependent control of expression at imprinted loci. Cell Stem Cell. 2011;9(3):272–281.

16. Ferron SR, Charalambous M, Radford E, et al. Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis. Nature. 2011;475(7356):381–385.

17. Li W, Sun W, Zhang Y, et al. Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors. Proc Natl Acad Sci U S A. 2011;108(20):8299–8304.

18. Liu GH, Qu J, Suzuki K, et al. Progressive degeneration of human neural stem cells caused by pathogenic LRRK2. Nature. 2012;491(7425):603–607.


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Stem Cell Therapy for Coronary Artery Disease (CAD)

Author and Curator: Larry H. Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN


There is great interest and future promise for stem cell therapy in ischemic heart disease.  This is another report for the active work in cardiology with stem cell therapy by MA Gaballa and associates at University of Arizona.

Stem Cell Therapy for Coronary Heart Disease

Julia N. E. Sunkomat and Mohamed A. Gaballa

The University ofArizona Sarver Heart Center, Section of Cardiology, Tucson, Ar
Cardiovascular Drug Reviews 2003: 21(4): 327–342

Keywords: Angiogenesis — Cardiac therapy — Coronary heart disease — Heart failure — Myoblasts — Myocardial ischemia — Myocardial regenera­tion — Stem cells


Coronary artery disease (CAD) remains the leading cause of death in the Western world. The high impact of its main sequelae, acute myocardial infarction and congestive heart failure (CHF), on the quality of life of patients and the cost of health care drives the search for new therapies. The recent finding that

stem cells contribute to neovascularization and possibly improve cardiac function after myocardial infarction makes stem cell therapy the most highly active research area in cardiology. Although the concept of stem cell therapy may revolutionize heart failure treatment, several obstacles need to be ad­dressed. To name a few:

  1.  Which patient population should be considered for stem cell therapy?
  2.  What type of stem cell should be used?
  3.  What is the best route for cell de­livery?
  4.  What is the optimum number of cells that should be used to achieve functional effects?
  5.  Is stem cell therapy safer and more effective than conventional therapies?

The published studies vary significantly in design, making it difficult to draw conclusions on the efficacy of this treatment. For example, different models of

  1. ischemia,
  2. species of donors and recipients,
  3. techniques of cell delivery,
  4. cell types,
  5. cell numbers and
  6. timing of the experiments

have been used. However, these studies highlight the landmark concept that stem cell therapy may play a major role in treating cardiovascular diseases in the near future. It should be noted that stem cell therapy is not limited to the treatment of ischemic cardiac disease.

  • Non-ischemic cardiomyopathy,
  • peripheral vascular disease, and
  • aging may be treated by stem cells.

Stem cells could be used as vehicle for gene therapy and eliminate the use of viral vectors. Finally, stem cell therapy may be combined with phar­macological, surgical, and interventional therapy to improve outcome. Here we attempt a systematic overview of the science of stem cells and their effects when transplanted into ischemic myocardium.



Congestive heart failure (CHF) is the leading discharge diagnosis in patients over the age of 65 with estimates of $24 billion spent on health care in the US (1,11). The number one cause of CHF is coronary artery diseases (CAD). Coronary care units, reperfusion therapy (lytic and percutaneous coronary intervention) and medical therapy with anti-pla­telet agents, statins, ACE-inhibitors and â-adrenoceptor antagonists all significantly reduce morbidity and mortality of CAD and CHF (9), but it is very difficult to regenerate new viable myocardium and new blood vessels.

Identification of circulating endothelial progenitor cells in peripheral blood that incor­porated into foci of neovascularization in hindlimb ischemia (4) and the successful engraftment of embryonic stem cells into myocardium of adult dystrophic mice (31) intro­duced a new therapeutic strategy to the field of cardiovascular diseases: tissue regeneration. This approach is supported by the discovery of primitive cells of extracardiac origin in cardiac tissues after sex-mismatched transplants suggesting that an endogenous repair mechanism may exist in the heart (35,45,54). The number of recruited cells varied significantly from 0 (19) to 18% (54), but the natural course of ischemic cardiomyopathy implies that cell recruitment for tissue repair in most cases is insufficient to prevent heart failure. Therefore, investigational efforts are geared towards

  • augmenting the number of multipotent stem cells and endothelial and myocardial progenitor cells at the site of ischemia to induce clinically significant angiogenesis and potentially myogenesis.

Stem and Progenitor Cells

Stem cells are defined by their ability to give rise to identical stem cells and progenitor cells that continue to differentiate into a specific tissue cell phenotype (23,33). The po­tential of mammalian stem cells varies with stage of development and age (Table 1).

In mammals, the fertilized oocyte and blastomere cells of embryos of the two to eight cell stage can generate a complete organism when implanted into the uterus; they are called totipotent stem cells. After the blastocyst stage, embryonic stem cells retain the ability to differentiate into all cell types, but

  • cannot generate a complete organism and thus are denoted pluripotent stem cells.

Other examples of pluripotent stem cells are embryo­nic germ cells that are derived from the gonadal ridge of aborted embryos and embryonic carcinoma cells that are found in gonadal tumors (teratocarcinomas) (23,33). Both these cell types can also differentiate into cells of all three germ layers, but are not as well inves­tigated as embryonic stem cells.

It is well established that embryonic stem cells can differentiate into cardiomyocytes (7,10,13,14,31,37,76), endothelial cells (55), and smooth muscle cells (5,22,78) in vitro, but it is unclear whether

  • pure populations of embryonic stem cell-derived cardiomyocytes can integrate and function appropriately in the heart after transplantation.
  • one study reported arrhythmogenic potential of embryonic stem cell-derived cardiomyocytes in vitro (80).

Adult somatic stem cells are cells that have already committed to one of the three germ layers: endoderm, ectoderm, or mesoderm (76). While embryonic stem cells are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is still unknown. The primary role of adult stem cells in a living organism is thought to be maintaining and repairing the tissue in which they reside. They are the source of more identical stem cells and cells with a progressively more distinct phenotype of specialized tissue cells (progenitor and precursor cells) (Fig. 1). Until recently adult stem cells were thought to be lineage-specific, meaning that they can only differentiate into the cell-type of their original tissue. This concept has now been challenged with the discovery of multipotent stem and progenitor cells (26, 50, 51).

The presence of multipotent stem and progenitor cells in adult mammals has vast im­plications on the availability of stem cells to research and clinical medicine. Recent publi­cations, however, have questioned whether the adaptation of a phenotype in those dogma-challenging studies is really a result of trans-differentiation or rather a result of cell and nuclear fusion (60,68,75,79). Spontaneous fusion between mammalian cells was first re­ported in 1961 (8), but how frequently fusion occurs and whether it occurs in vivo is not clear.

The bone marrow is a known source of stem cells. Hematopoietic stem cells are fre­quently used in the field of hematology. Surface receptors are used to differentiate hematopoietic stem and progenitor cells from mature cells. For example, virtually all

  • hematopoietic stem and progenitor cells express the CD34+ glycoprotein antigen on their cell membrane (73),

though a small proportion of primitive cells have been shown to be CD34 negative (58).

The function of the CD34+ receptor is not yet fully understood. It has been suggested that it may act as a regulator of hematopoietic cell adhesion in the bone marrow microenvironment. It also appears to be involved in the maintenance of the hematopoietic stem/progenitor cell phenotype and function (16,21). The frequency of immature CD34+ cells in peripheral circulation diminishes with age.

  • It is the highest (up to 11%) in utero (69) and decreases to 1% of nucleated cells in term cord blood (63).
  • This equals the per­centage of CD34+ cells in adult bone marrow.
  • The number of circulating stem cells in adult peripheral blood is even lower at 0.1% of nucleated cells.

Since hematopoietic stem cells have been identified as endothelial progenitor cells (29,30,32) their low density in adult bone marrow and blood could explain the inadequacy of endogenous recruitment of cells to injured organs such as an ischemic heart. The bone marrow is also home to another stem cell population the so-called mesenchymal stem cells. These may constitute a subset of the bone marrow stromal cells (2,43). Bone marrow stromal cells are a mixed cell popu­lation that generates

  1. bone,
  2. cartilage,
  3. fat,
  4. connective tissue, and
  5. reticular network that sup­ports cell formation (23).

Mesenchymal stem cells have been described as multipotent (51,52) and as a source of myocardial progenitor cells (41,59). They are, however, much less defined than the hematopoietic stem cells and a characteristic antigen constellation has not yet been identified (44).

Another example of an adult tissue containing stem cells is the skeletal muscle. The cells responsible for renewal and growth of the skeletal muscle are called satellite cells or myoblasts and are located between the sarcolemma and the basal lamina of the muscle fiber (5). Since skeletal muscle and cardiac muscle share similar characteristics such as they both are striated muscle cells, satellite cells are considered good candidates for the repair of damaged myocardium and have been extensively studied (20,25,38–40,48,56, 64–67). Myoblasts are particularly attractive, because they can be autotransplanted, so that issues of donor availability, ethics, tumorigenesis and immunological compatibility can be avoided. They also have been shown to have a high growth potential in vitro and a strong resistance to ischemia in vivo (20). On the down side

  • they may have more arrhythmogenic potential when transplanted into myocardium than bone marrow or peripheral blood de­rived stem cells and progenitor cells (40).

Isolation of Cells Prior to Transplantation

Hematopoietic stem and progenitor cells are commonly identified by the expression of a profile of surface receptors (cell antigens). For example, human hematopoietic stem cells are defined as CD34+/CD59+/Thy-1+/CD38low//c-kit/low/lin, while mouse hema-topoietic stem cells are defined as CD34low//Sca-1+/Thy-1+/low/CD38+/c-kit+/lin (23). Additional cell surface receptors have been identified as markers for subgroups of hema-topoietic stem cells with the ability to differentiate into non-hematopoetic tissues, such as endothelial cells (57,78). These can be specifically targeted by isolation methods that use the receptors for cell selection (positive selection with antibody coated magnetic beads or fluorescence-activated cell sorting, FACS). Other stem cell populations are identified by their behavior in cell culture (mesenchymal stem cells) or dye exclusion (SP cells). Finally, embryonic stem cells are isolated from the inner cell mass of the blastocyst and skeletal myoblasts are mechanically and enzymatically dissociated from an easily acces­sible skeletal muscle and expanded in cell culture.

FIG. 1. Maturation process of adult stem cells: with acquisition of a certain phenotype the cell gradually loses its self-renewal capability.  (unable to transfer)


j.1527-3466.2003.tb00125.x  fig stem cell

FIG. 2. Intramyocardial injection:

the cells are injected directly into the myocardium through the epicardium. Usually a thoracotomy or sternotomy is required. Transendocardial injection: access can be gained from the ar­terial vasculature. Cells are injected through the endocardium into the myocardium, ideally after identifying the ischemic myocardium by perfusion studies and/or electromechanical mapping. Intracoronary injection: the coronary artery is accessed from the arterial vasculature. Stem cells are injected into the lumen of the coronary artery. Proximal washout is prevented by inflation of a balloon. Cells are then distributed through the capillary system. They eventually cross the endothelium and migrate towards ischemic areas.

The intracoronary delivery of stem cells (Fig. 2) and distribution through the coronary system has also been explored (6,62,74). This approach was pioneered by Robinson et al. (56), who demonstrated successful engraftment within the coronary distribution after intracoronary delivery of genetically labeled skeletal myoblasts. The risk of intracoronary injection is comparable to that of a coronary angiogram and percutaneous transluminal coronary angioplasty (PTCA) (62), which are safe and clinically well established.


Dif­ferentiation into cardiomyocytes was observed after transplantation of embryonic stem cells, mesenchymal stem cells, lin/c-kit+ and SP cells. The induction of angiogenesis was observed after transplantation of embryonic stem cells, mesenchymal stem cells, bone marrow-derived mononuclear cells, circulating endothelial progenitor cells, SP cells and lin/c-kit+ cells.

The use of embryonic stem cells in ischemia was examined in two studies (42,43). These studies demonstrated that mice embryonic stem cells transplanted into rat myo­cardium exhibited cardiomyocyte phenotype at 6 weeks after transplantation. In addition, generation of myocardium and angiogenesis were observed at 32 weeks after allogenic transplantation in rats. In these two studies no arrhythmias or cardiac tumors were reported.

Several studies have shown retardation of LV remodeling and improvement of cardiac function after administration of bone marrow-derived mononuclear cells. For example, decreases in infarct size, and increase in ejection fraction (EF), and left ventricular (LV) time rate change of pressure (dP/dtmax) were observed after direct injection of bone marrow-derived mononuclear cells 60 min after ischemia in swine (28). In humans, intra-coronary delivery and transendocardial injection of mononuclear cells leads to a decrease in LV dimensions and improvement of cardiac function and perfusion (49,62). A decrease in end systolic volume (ESV) and an increase in EF as well as regional wall motion were observed following intracoronary administration of CD34+/CD45+ human circulating en­dothelial cells (6). Injection of circulating human CD34+/CD117+ cells into infarcted rat myocardium induced neoangiogenesis and improved cardiac function (32). This study suggests that the improvement in LV remodeling after infarction appears to be in part me­diated by a decrease in apoptosis within the noninfarcted myocardium. Two other studies reported increased fractional shortening, improved regional wall motion and decreased left ventricular dimensions after transplantation of human CD34+ cells (29,30). Improved global left ventricular function and infarct perfusion was demonstrated after intramyo-cardial injection of autologous endothelial progenitor cells in humans (61).


The idea of replacing damaged myocardium by healthy cardiac tissue is exciting and has received much attention in the medical field and the media. Therefore, it is important for the scientist to know what is established and what is based on premature conclusions. Currently, there are data from animal studies and human trials (Table 2). However, some of these data are not very concrete. For example,

  • many animal studies do not report the level of achieved neoangiogenesis and/or regeneration of myocardium.
  • In studies where the numbers of neovessels and new cardiomyocytes are specified, these numbers are often very low.

While these experiments confirm the concept that bone marrow and peripheral blood-derived stem and progenitor cells can differentiate into cardiomyocytes and endo­thelial cells when transplanted into ischemic myocardium, they also raise the question how effective this treatment is.

The results of the clinical trials that have been conducted are encouraging, but they need to be interpreted with caution. The common endpoints of these studies include left ventricular dimensions, perfusion, wall motion and hemodynamic function. While all studies report improvement after mononuclear cell, myoblast or endothelial progenitor cell transplantation, it is difficult to separate the effects of stem cell transplantation from the effects of the state-of-the art medical care that the patients typically received.


While the majority of studies demonstrate neoangiogenesis and some studies also show regeneration of myocardium after stem/progenitor cell transplantation, it remains unclear whether the currently achieved level of tissue regeneration is sufficient to affect clinical outcome. Long-term follow-up of patients that received stem/progenitor cells in clinical trials will provide important information on the potential risks of neoplasm and arrhythmias and, therefore, safety of this treatment. Ultimately, postmortem histological confirmation of scar tissue repair by transplanted cells and randomized placebo control trials with long-term follow-up are required to prove efficacy of this treatment.


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2. Arai A, Sheikh F, Agyeman K, et al. Lack of benefit from cytokine mobilized stem cell therapy for acute myocardial infarction in nonhuman primates. J Am Coll Cardiol 2003;41(Suppl 6A):371.

3. Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999;85:221–228.

4. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.

5. Asakura A, Seale P, Girgis-Gabardo A, Rudnicki M. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159(1):123–134.

6. Assmus B, Schaechinger V, Teupe C, et al. Transplantation of progenitor cells and regeneration en­hancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106:r53–r61.

7. Bader A, Al-Dubai H, Weitzer G. Leukemia inhibitory factor modulates cardiogenesis in embryoid bodies in opposite fashions. Circ Res 2000;86(7):787–794.

8. Barski G, Sorieul S, Cornefert F. “Hybrid” type cells in combined cultures of two different mammalian cell strains. J Natl Cancer Inst 1961;26:1269–1291.

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Author and Curator: Ritu Saxena, PhD


What are cancer stem cells?

Cancer is a debilitating disease estimated to be responsible for about 7.6 million deaths in 2008 (Jemal A, et al, CA Cancer J Clin, Mar-Apr 2011;61(2):69-90). Thus, extensive research is underway to deal with the various types of cancer. The concept of cancer stem cells (CSC) has surfaced in in the past decade after identification and characterization of CSC-enriched populations in several different types of cancer (Lapidot T, et al, Nature, 17 Feb 1994;367(6464):645-8; Reya T, et al, Nature, 1 Nov 2001;414(6859):105-11;  Trumpp A and Wiestler OD, et al, Nat Clin Pract Oncol, Jun 2008;5(6):337-47). Although there has been lot of debate on the cell of origin of CSC, according to the classical concept CSC are defined by their functional properties.

Functional properties of CSC

  • CSCs are at the top of tumor hierarchy. Regenerative tissues follow a hierarchical organization with adult stem cells at the top maintaining tissues and normal adult cells during homeostasis and regeneration during cell loss from injury. Similarly, several tumors follow the hierarchy with CSC at the top. Hierarchical organization has been reported in several cancer types including but not limited to breast cancer, brain cancer, colon cancer, leukemia and pancreatic cancer (Lapidot T, et al, Nature, 17 Feb 1994;367(6464):645-8; Al-Hajj M, et al, PNAS USA, 1 Apr 200;100(7):3983-8; Singh SK, et al, Nature, 18 Nov 2004;432(7015):396-401; Dalerba P, et al, PNAS USA, 12 Jun 2007;104(24):10158-63; Hermann PC, et al, Cell Stem Cell, 13 Sep 2007;1(3):313-23).
  • CSCs possess unlimited self-renewal capacity similar to that of physiological stem cells and unlike other differentiated cell types within the tumor. Cancer stem cells can also generate non-CSC progeny that is comprised of differentiated cells and forms tumor bulk.
  • Some CSs exhibit quiescent or dormant stage. Although not observed in all CSC types, some CSCs have been found to shuttle between quiescent, slow-cycling, and active states. The CSCs in their dormant and slow-cycling stage are less likely to be affected by conventional anti-tumor therapies which generally target rapidly dividing cells. Dormant stage is exhibited even in adult stem cells and the dormant normal stem cells can regain cell division potential during tissue injury (Wilson A, et al, Cell,  12 Dec 2008;135(6):1118-29). Thus, it has been speculated that dormant CSC might be a reason for tumor relapse even after pathologic complete response is observed post therapy.
  • Some CSCs are resistant to conventional anti-cancer therapies. This leads to accumulation of CSC that might result in relapse after anti-cancer therapy. For instance, Li et al (2008) reported that CSC accumulated in the breast of women with locally advanced tumors after cytotoxic chemotherapy had eliminated the bulk of the tumor cells (Li X,et al, J Natl Cancer Inst, 7 May 2008;100(9):672-9). A similar observation was made by Oravecz-Wilson et al (2009) stating that despite remarkable responses to the tyrosine kinase inhibitor imatinib, CML patients show imatinib refractoriness because leukemia stem cells in CML are resistant tyrosine kinase (Oravecz-Wilson KI, et al, Cancer Cell, 4 Aug 2009;16(2):137-48).
  • The CSC niche. CSC functional traits might be sustained by this microenvironment, termed “niche”. The niche is the environment in which stem cells reside and is responsible for the maintenance of unique stem cell properties such as self-renewal and an undifferentiated state. The heterogeneous populations which constitute a niche include both stem cells and surrounding differentiated cells. The necessary intrinsic pathways that are utilized by this cancer stem cell population to maintain both self-renewal and the ability to differentiate are believed to be a result of the environment where cancer stem cells reside. (Cabarcas SM, et al, Int J Cancer, 15 Nov 2011;129(10):2315-27). For instance, properties of CSC in glioma in a mouse xenograft model were maintained by vascular endothelial cells (Calabrese C, et al, Cancer Cell, Jan 2007;11(1):69-82). Several molecules including interleukin 6 have been observed to play a role in tumor proliferation and hence, participate in maintaining tumorigenic and self-renewal potential of CSC. Moreover, the CSC niche might not only regulate CSCs traits but might also directly provide CSC features to non-CSC population.

What is the origin of CSC?

According to current thinking, CSC result from epithelial-mesenchymal transition (EMT) when cells switch from a polarized epithelial to a non-polarized mesenchymal cell type with stem cell properties, including migratory behavior, self-renewal and generation of differentiated progeny, and reduced responsiveness to conventional cancer therapies (Scheel C and Weinberg RA, Semin Cancer Biol, Oct 2012;22(5-6):396-403; Crews LA and Jamieson CH, Cancer Lett, 17 Aug 2012). Evidence is accumulating that cancers of distinct subtypes within an organ may derive from different ‘cells of origin’. The tumor cell of origin is the cell type from which the disease is derived after it undergoes oncogenic mutation. It might take a series of mutations to achieve the CSC phenotype (Visvader JE, Nature, 20 Jan 2011;469(7330):314-22). Also, CSCs have been reported to originate from stem cells in some cases.

Biomarkers for CSC

CSC targeting therapy could either eliminate CSCs by either killing them after differentiating them from other tumor population, and/or by disrupting their niche. Efficient eradication of CSCs may require the combined ablation of CSCs themselves and their niches. Identifying appropriate biomarkers of CSC is a very important aim for CSCs to be useful as targets of anti-cancer therapies in order to possibly prevent relapse. Using cell surface markers, CSCs have been isolated and purified from cancers of breast, brain, thyroid, cervix, lung, blood (leukemia), skin (melanoma), organs of the gastrointestinal and reproductive tracts, and the retina. The challenge, however, is that CSCs share similar markers with normal cells which makes CSCs targeting difficult as it would harm normal cells in the process. More recently, advanced techniques such as signal sequence trap (SST) PCR screening methods have been developed to identify a leukemia-specific stem cell marker (CD96). After a small subset of human AML cells displayed tumorigenic properties, Leukemia Stem Cells (LSCs) were identified as leukemia cells with CD23+/CD38+ markers. These cells closely resemble hematopeotic stem cells (HSCs) (Bonnet D and Dick JR, Nat Med, Jul 1997;3(7):730-7). In solid tumors, a significant discovery was made when CSCs in breast cancer were identified within the ESA+/CD44+/CD24low-neg population of mammary pleural effusion and tumor samples (Al-Hajj M, et al, PNAS USA, 1 Apr 200;100(7):3983-8).

After these two landmark publications, CSCs were identified in many more solid and hematopoietic human tumors as well. In addition, within a tumor type, CSC-enriched populations display heterogeneity in markers. For example, only 1% of breast cancer cells simultaneously express both reported CSC phenotypes ESA+/CD44+/

CD24low-neg and ALDH-1+ (Ginestier C, et al, Cell Stem Cell, 1 Nov 2007;1(5):555-67). The discrepancy might be due to different techniques used to identify the markers and also a reflection of the molecular heterogeneity within the tumors. Recent advances in genome wide expression profiling studies have led to the identification of different subtypes in a particular type of cancer. Breast cancer was recently classified into different subtypes and this genetic heterogeneity is likely paralleled by a heterogeneous CSC complexity.


A lot of research is currently underway on various aspects of CSCs including biomarker identification, cell of origin, and clinical trials targeting CSC population in cancer. The concept of CSCs has evolved quite a bit since their discovery. Recently, identification of high genetic heterogeneity within a tumor has been in focus and subsequently it has been observed that several CSC clones can coexist and compete with each other within a tumor. Adding complexity to their identity is the fact that CSCs may have unstable phenotypes and genotypes. Taken together, the dynamics associated with CSCs makes it difficult to identify reliable and robust biomarkers and develop efficient targeted therapies. Thus, a major thrust of research should be to focus on the unfolding of the dynamic identity of CSCs in tumor types and at different that might lead to the identification and targeting of highly specific CSCs biomarkers.


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Pharmaceutical Intelligence posts:

Authors: Anamika Sarkar, PhD and Ritu Saxena, PhD Author: Ziv Raviv, PhD Reporter: Larry H Bernstein, MD

Larry H Bernstein, MD Curator: Aviva Lev-Ari, PhD, RN Curator: Ritu Saxena, PhD Curator: Aviva Lev-Ari, PhD, RN Author and reporter: Tilda Barliya PhD Reporter and Curator: Stephen J. Williams, PhD Reporter: Ritu Saxena, PhD Reporter: Aviva Lev-Ari, PhD, RN Reporter: Ritu Saxena, PhD Aviva Lev-Ari, PhD, RN

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Author: Tilda Barliya PhD

Acute lymphoblastic leukemia (ALL), a malignant disorder of lymphoid progenitor cells, affects both children and adults,
with peak prevalence between the ages of 2 and 5 years (2). Acute lymphocytic leukemia (ALL) is a heterogeneous disease, both in terms of its pathology and the populations that it affects. Disease pathogenesis involves a number of deregulated pathways controlling cell proliferation, differentiation, and survival that are important determinants of treatment response (3). Approximately 5200 new cases of ALL are estimated to have occurred in the United States in 2007 and survival varies with age and disease biology (3). Although five-year survival rates for ALL approach 90 percent with available chemotherapy treatments, the harmful side effects of the drugs, including secondary cancers and fertility, cognitive, hearing, and developmental problems, present significant concern for survivors and their families.

Biological and Clinical Prognostic Factors in ALL: Setting the Stage for Risk-Adapted Therapy

Of the many variables that influence prognosis the genetic subsets, initial white blood cell count (WBC), age at diagnosis, and early treatment response are the most important.

Childhood Acute Lymphoblastic Leukemia


Acute lymphoblastic leukaemia is thought to originate  from various important genetic lesions in blood-progenitor  cells that are committed to differentiate in the T-cell or B-cell pathway, including mutations that impart the  capacity for unlimited self-renewal and those that lead to  precise stage-specific developmental arrest. In some  cases, the first mutation along the multistep pathway to  overt acute lymphoblastic leukaemia might arise in a  haemopoietic stem cell possessing multilineage developmental capacity.

The dominant theme of contemporary research in pathobiology of acute lymphoblastic leukaemia is to understand the outcomes of frequently arising genetic lesions, in terms of their effects on cell proliferation, differentiation, and survival, and then to devise selectively targeted treatments against the altered gene products to which the leukaemic clones have become addicted (2).

Table 1.

Prognostic factors used in pediatric and adult clinical trials

The Table  illustrates the different prognostic factors in children and adults that may be used for risk stratification in current clinical trials (3).


  • Chromosomal translocations that activate specifi c genes
    are a defi ning characteristic of human leukaemias and
    of acute lymphoblastic leukaemia in particular.
  • About 25% of cases of B-cell precursor acute lymphoblastic leukaemia, the most frequent form of acute leukaemia in children, harbour the TEL-AML1 fusion gene—generated by the t(12;21)(p13;q22) chromosomal translocation.

The presence of the TEL-AML1 fusion
protein in B-cell progenitors seems to lead to disordered
early B-lineage lymphocyte development, a hallmark of
leukaemic lymphoblasts.

Analysis of TEL-AML1-induced cord blood cells suggests that the fusion gene serves as a first-hit mutation by endowing the preleukemic cell with altered self-renewal and survival properties.

  • In adults, the most frequent chromosomal translocation  is t(9;22), or the Philadelphia chromosome, which causes  fusion of the BCR signalling protein to the ABL  non-receptor tyrosine kinase, resulting in constitutive  tyrosine kinase activity and complex interactions of this  fusion protein with many other transforming elements.  BCR-ABL off ers an attractive therapeutic  target, and imatinib mesilate, a small-molecule inhibitor  of the ABL kinase, has proven effective against leukaemias that express BCR-ABL
  • More than 50% of cases of T-cell acute lymphoblastic  leukaemia have activating mutations that involve  NOTCH1. NOTCH1, which translocates to the nucleus and regulates by transcription a diverse set of responder genes, including the MYC oncogene.  The precise  mechanisms by which aberrant NOTCH signalling (due  to mutational activation) causes T-cell acute lymphoblastic  leukaemia are still unclear but probably entail constitutive  expression of oncogenic responder genes, such as MYC,  and cooperation with other signalling pathways (pre-TCR  [T-cell receptor for antigen] and RAS, for example).  Interference with NOTCH signalling by small-molecule  inhibition of γ-secretase activity has the potential to induce remission of T-cell acute lymphoblastic  leukemia.

Additionally A recent discussion has aimed to reveal the genetic origin of the disease (1). Several of these genes, including ARID5B, IKZF1, and CEBPE, have been implicated in processes such as hematopoietic differentiation and development of ALL. These gene obviously adds up to a number of other gene mutations and translocation already discovered and are associated with disease progression (2)  “The fact that alterations in these genes lead to ALL raises the question of what would happen if we restore these pathways in ALL and also make them possible exciting therapeutic targets as well.”

Nanotechnology and therapeutic

Dr. Rajasekaran, director and head of the Membrane Biology Laboratory University of Delaware,  says that there are currently seven or eight drugs that are used for chemotherapy to treat leukemia in children. They are all toxic and do their job by killing rapidly dividing cells. these drugs don’t differentiate cancer cells from other healthy cells. “The good news is that these drugs are 80 to 90 percent effective in curing leukemia. The bad news is that many chemotherapeutic treatments cause severe side effects, especially in children.  In preclinical models of leukemia, Dr. Rajasekaran research team have created NP  with an average diameter of 110 nm were assembled from an amphiphilic block copolymer of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) bearing pendant cyclic ketals (ECT2). The researches have been encapsulated with dexamethasone as one third of the typical dose, with good treatment results and no discernible side effects.In addition, the mice that received the drugs delivered via nanoparticles survived longer than those that received the drug administered in the traditional way (4).

In another preclinical study Uckun F et al  developed nanoparticle (NP) constructs of WHI-P131. WHI-P131 (CAS 202475-60-3) is a dual-function inhibitor of JAK3 tyrosine kinase that demonstrated potent in vivo anti-inflammatory and anti-leukemic activity in several preclinical animal models (5). Notably, WHI-P131-NP was capable of causing apoptotic death in primary leukemia cells from chemotherapy-resistant acute lymphoblastic leukemia (ALL) as well as chronic lymphocytic leukemia (CLL) patients. WHI-P131-NP was also active in the RS4;11 SCID mouse xenograft model of chemotherapy-resistant B-lineage ALL. The life table analysis showed that WHI-P131-NP was more effective than WHI-P131 (P = 0.01), vincristine (P<0.0001), or vehicle (P<0.0001). These experimental results demonstrate that the nanotechnology-enabled delivery of WHI-P131 shows therapeutic potential against leukemias with constitutive activation of the JAK3-STAT3/STAT5 molecular target (5).


Acute Lymphoblastic Leukemia (ALL) is a pediatric type of cancer that affects adults to lesser degree. The current rate of cure if 80% in  children whereas in adults only 30-40% will survive. Much of the success is due to understanding the mechanisms that lead to the development and progression of cancer. Several gene mutations and gene-translocation have already been identified,  and targeting them enabled some of the major success in curing these kids.

Thus far, nanotechnology has been  mainly focusing on solid tumors affecting adults. Not much attention is been made on childhood cancer in general and hematopoietic types per se. Two examples of preclinical studies have been discussed above and although they show promise in treatment and reduction of side effects, yet  additional research is needed to evaluated their effect in human clinical trials.


1. The Genetic Origin of Childhood Acute Lymphoblastic Leukemia (ALL).  Reported by Aviva Lev-Ari, PhD, RN. March 20, 2013

2. Ching-Hon Pui, Leslie L Robison, A Thomas Look. Acute lymphoblastic leukaemia. Lancet 2008; 371: 1030–43.

3. Wendy Stock. Adolescents and Young Adults with Acute Lymphoblastic Leukemia. Hematology December 4, 2010 vol. 2010 no. 1 21-29.

4. Vinu Krishnan,  Xian Xu,, Sonali P. BarweXiaowei YangKirk CzymmekScott A. WaldmanRobert W. MasonXinqiao Jia, and Ayyappan K. Rajasekaran. Dexamethasone-Loaded Block Copolymer Nanoparticles Induce Leukemia Cell Death and Enhance Therapeutic Efficacy: A Novel Application in Pediatric Nanomedicine. Mol. Pharmaceutics 2012 ahead of print.

5. Uckun FMDibirdik IQazi SYiv S. Therapeutic nanoparticle constructs of a JAK3 tyrosine kinase inhibitor against human B-lineage ALL cells. Arzneimittelforschung 2010; 60(4): 210-217.

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