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Archive for the ‘Nephrology & Regenerative medicine’ Category


Stem Cell derived kidneys

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

HUMAN STEM CELL-DERIVED KIDNEYS CONNECT TO BLOOD VESSELS WHEN TRANSPLANTED INTO MICE

http://health-innovations.org/2015/11/20/human-stem-cell-derived-kidneys-connect-to-blood-vessels-when-transplanted-into-mice/

 

ft Stem cell-derived kidneys connect to blood vessels when transplanted into mice - healthinnovations

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The kidney tissues derived from human iPS cells
A.The kidney tissue generated in vitro, which shows green fluorescence in each glomerulus.
B.Vascularized glomerulus formed upon transplantation into the mouse. Many red blood cells (arrowhead) are observed, and the substance exists in the lumen (*), suggesting the possible filtration.
C.Mouse vascular endothelial cells (green) are incorporated into the glomerulus that consists of podocytes (magenta).
D.The slit diaphragm (arrow) formed between the cellular processes of the podocytes. Credit: The Institute of Molecular Embryology and Genetics (IMEG).

In the field of iPS cell-based regenerative medicine, advanced research with clinical applications for many organs and tissues, such as the retina, has steadily progressed. However, growing a kidney from scratch has been extremely difficult.  Although the number of renal failure patients on dialysis is increasing, opportunities for renal transplant have been limited with great attention given to the growth of kidneys to stem the shortage.

Now, a study from researchers at Kumamoto University shows mouse kidney capillaries successfully connecting to kidney tissue derived from human iPS cells. The team state that this achievement shows that human kidney glomeruli made in vitro can connect to blood vessels after transplantation and grow to maturity, a big step forward in gain-of-function for a urine-producing kidney.  The opensource study is published in the Journal of the American Society of Nephrology.

Earlier studies from the lab led to the development of an in vitro three-dimensional kidney structure from human iPS cells.  However, it was unclear how similar the kidney tissue made in vitro was to that formed in a living body. Additionally, the original kidney tissue was not connected to any blood vessels, even though the primary function of the organ is to filter waste products and excess fluid from the blood.  In many kidney diseases, the pathology is with the glomeruli that filter urine from the blood. Filtration in the glomerulus is performed by cells called podocytes that are in direct contact with the blood vessels. Through the special filtration membrane of the podocytes, proteins don’t leak into the urine and allows moisture to pass through.  Therefore, the group focused on analyzing the podocyte of the glomeruli in detail.  They achieved this by genetically modifying the iPS cells and growing human kidney tissue in vitro with green fluorescence then visualizing how human glomeruli became established.

The current study continued this analysis by taking out only the podocytes of the human glomeruli using the green fluorescence, and revealed that glomerular podocytes made in vitro express the same genes important for normal biological function.  Data findings show that after transplanting the human iPS cell-based kidney tissue into a mouse body, glomeruli connecting to mouse kidney capillaries formed. Results show that human glomerular podocytes further matured around adjacent blood vessels as in a living body and formed a characteristic filtration membrane structure.  The group state that to their knowledge the successful connection of capillaries with the podocytes of iPS cell-manufactured human glomeruli resulting in a distinct filtration membrane is the first of its kind in the world.

The team surmise that their findings should advance research into the manufactured kidney’s function to produce and excrete urine.  They go on to add that by using iPS cells from patients, development of new drugs and clarification of the causes of kidney disease are also expected.  For the future, the researchers state that they are now working to develop a discharge path for the kidney and combine it with findings on glomerular cells.

Source: The Institute of Molecular Embryology and Genetics (IMEG)

 

Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation

Sazia Sharmin*Atsuhiro Taguchi*Yusuke Kaku*Yasuhiro Yoshimura*Tomoko Ohmori*Tetsushi Sakuma, et al.

JASN Nov 19; 2015 ASN.2015010096      http://dx.doi.org:/10.1681/ASN.2015010096    http://jasn.asnjournals.org/content/early/2015/11/18/ASN.2015010096.full

Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro. These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm–like structures. Microarray analysis of sorted iPS cell–derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo. Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell–derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell–derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases.

 

The glomerulus is the filtering apparatus of the kidney and contains three types of cells: podocytes, vascular endothelial cells, and mesangial cells. Podocytes cover the basal domains of the endothelial cells via the basement membrane and play a major role in the filtration process.1,2 Podocytes possess multiple cytoplasmic protrusions. The primary processes are complicated by the further stemming of smaller protrusions (secondary processes or foot processes), which interdigitate with those from neighboring podocytes. The gaps between these foot processes are connected with the slit diaphragm, which is detectable only by electron microscopy. The molecular nature of the slit diaphragm was initially revealed by identification of NPHS1 as the gene responsible for Finnish-type congenital nephrotic syndrome.3 The nephrin protein encoded by NPHS1intercalates with those from neighboring cells, thus forming a molecular mesh that hinders serum proteins of high molecular weight from leaking into the urine.4,5 To date, many slit diaphragm–associated proteins have been identified, including NPHS2 (encoding podocin) and NEPH1, mutations that cause proteinuria in humans and/or mice.6,7

Podocytes are derived from nephron progenitors that reside in the embryonic kidney and express transcription factor Six2.8 Upon Wnt stimulation, the nephron progenitors undergo mesenchymal-to-epithelial transition and form a tubule.9 This tubule changes its shape; one end forms the glomerulus with podocytes inside, which is surrounded by a Bowman’s capsule. Meanwhile, vascular endothelial cells and mesangial cells migrate into the developing glomeruli, thus connecting the glomeruli with circulation.2 In these processes, several transcription factors, including Wt1, regulate expression of nephrin in podocytes.10 Apical junctions are initially formed between the presumptive podocytes, but the apical domain loses its direct contact with that of the neighboring cells, thus forming the characteristic podocyte shape. Nephrin is eventually localized to the site close to the basal domain and contributes to the formation of the slit diaphragm.2 The molecular mechanisms underlying podocyte development have been extensively studied in mice. However, because of limited access to human embryos, relatively little is known regarding transcription profiles of podocytes and glomerulogenesis in humans.4,11,12

We have recently induced the nephron progenitors from mouse embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells by redefining the in vivo origin of the nephron progenitors.13 The induced progenitor aggregates readily form three-dimensional primordial glomeruli and renal tubules upon Wnt stimulation in vitro. To analyze the detailed structures and transcription profiles of the induced podocytes, we have here inserted the GFP gene into the NPHS1 locus of human iPS cells via homologous recombination using transcription activator–like effector nucleases (TALENs)14 and generated glomeruli with green fluorescent protein (GFP)-tagged podocytes.

 

Fluorescent Visualization of Human Glomerular Podocytes Generated fromNPHS1-GFP iPS Cells

To visualize developing human podocytes in vitro, we inserted a gene encoding GFP into the NPHS1 locus of human iPS cells by homologous recombination (Figure 1A). We first constructed a pair of plasmids expressing TALENs targeted in close proximity to the NPHS1 start codon. When tested in HEK 293 cells, these plasmids efficiently deleted the NPHS1 gene (Supplemental Figure 1A). We then introduced these TALEN plasmids, along with a targeting vector containing the GFP gene and the homology arms, into human iPS cells. This resulted in efficient homologous recombination and isolation of heterozygous GFP knock-in (NPHS1-GFP) clones (Figure 1B, Supplemental Figure 1, B and C).

Figure 1.

Successful generation ofNPHS1-GFP iPS cells by homologous recombination. (A) Strategy for targeting the human NPHS1 locus. TheGFP cassette was inserted upstream of the NPHS1 start codon. The puromycin-resistance cassette (PURO) is flanked by loxP sites. Positions for primers and probes for screening are indicated. E, EcoRV; N, NheI. (B) Southern blot of control (+/+) and NPHS1-GFP (GFP/+) clones. Genomic DNA was digested and hybridized with the indicated probes.

We differentiated these NPHS1-GFP iPS clones toward the nephron progenitors and subsequently combined them with murine embryonic spinal cord, which is a potent inducer of tubulogenesis, as we previously reported.13 Four days after recombination, spotty GFP signals could be observed, and the number and intensity of GFP signals increased thereafter until day 9 (Figure 2A,Supplemental Figure 2A). We observed GFP signals in all the examined samples from seven independent experiments (a total of 50 samples). Some of the signals started in a crescent shape and gradually changed into round structures (Figure 2A, lower panels), which suggests that human glomerular formation in vitro may be visualized. Therefore, we examined glomerulogenesis using sections of the explants. At day 3, only tubular structures were observed and GFP-positive cells were undetectable (Figure 2B). At day 4, structures that resembled S-shaped bodies were observed, in which proximo-distal specification occurred toward the presumptive distal (E-cadherin+) and proximal (cadherin-6+) renal tubules and glomerular podocytes (WT1+) (Figure 2C). At day 6, various forms of primordial glomeruli were observed, and most of the GFP signals overlapped with those of WT1 (Figure 2B). We ordered these glomeruli according to GFP intensity, which is likely to reflect the chronologic order of development. Weakly GFP-positive (and WT1-positive) limbs appeared at one end of the tubules, which elongated to surround the renal tubules. GFP intensity increased when the podocyte layers were convoluted. At day 9, strongly GFP-positive round glomeruli were formed. These histologic changes are consistent with the previous observations of human glomeruli in aborted fetuses.15 Thus, we succeeded in visualizing human podocyte development and glomerulogenesis in vitro. Interestingly, some, but not all, of the Bowman’s capsule cells were positive for GFP and nephrin (Supplemental Figure 2B), suggesting that these cells are not completely specified yet. Indeed, transient nephrin expression in some capsule cells was reported in vivo.16

Figure 2.

Fluorescent visualization of human glomerular podocytes generated fromNPHS1-GFP iPS cells. (A) Morphologic changes of GFP-positive glomeruli during differentiation in vitro. The nephron progenitors induced fromNPHS1-GFP iPS cells were combined with murine embryonic spinal cord and cultured for the indicated time. Lower panels: higher magnification of the areas marked by rectangles in the upper panels. Note the shape changes of the glomerulus (arrowheads). Scale bars: 500 μm. (B) Histologic sections of glomeruli developing in vitro. Tissues at day 3, 6, and 9 after recombination with the spinal cord were analyzed. Top panels: Hematoxylin-eosin (HE) staining. Middle panels: GFP (green) staining. Bottom panels: dual staining with GFP and WT1. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI: blue). Scale bars: 20 μm. (C) Presumptive S-shaped bodies observed at day 4 (left two panels) and day 6 (right two panels). Serial sections were stained with E-cadherin (Ecad: magenta)/cadherin-6 (cad6: green) and E-cadherin (magenta)/WT1 (green). Arrowheads: WT1-positive presumptive glomerular regions. Scale bars: 20 μm.

Induced Podocytes Exhibit Apicobasal Polarity and Basally Localized Nephrin

We analyzed day 9 sections at higher resolution to examine the apicobasal polarity of the induced podocytes. GFP was detected in the nuclei and cytoplasm of most cells in the round glomeruli (Figure 3A) because we did not attach any localization signal to GFP when generating NPHS1-GFP iPS cells. Nephrin proteins were distributed in a linear fashion in the iPS cell–derived glomeruli and at one end of the WT1-positive podocyte layer (Figure 3, A and B). These expression patterns significantly overlapped with those of type IV collagen, which was accumulated on the basal side of the podocytes (Figure 3C). In contrast, podocalyxin, an apical marker, was expressed in a manner mutually exclusive of nephrin (Figure 3D). Therefore, the induced podocytes exhibited a well established apicobasal polarity and nephrin proteins were properly localized at the basal side, where the presumptive slit diaphragm should be formed. We also observed nephrin-positive dots on the lateral side of the podocytes (Figure 3A, arrowheads), as reported in human developing podocytes in vivo.15 We found that these dots actually represent the filamentous structures encompassing the basal to the lateral side of the podocytes (Figure 3, B and C, arrowheads). Although further investigation is required, this may reflect the transit state of nephrin proteins shifting from the apical to the basal domain of the induced podocytes.

Figure 3.

Induced podocytes exhibit apicobasal polarity and basally localized nephrin. (A) Nephrin (magenta) and GFP (green) staining of the induced glomerulus at day 9. (B) Nephrin (magenta) and WT1 (green) staining. (C) Nephrin (magenta) and type IV collagen (COL: green) staining. (D) Nephrin (magenta) and podocalyxin (PODXL: green) staining. The left columns are at lower magnification to show a whole glomerulus. The right two columns are singly stained, while the left two columns represent merged images. Arrows: nephrin proteins localized to the basal domain; arrowheads: nephrin-positive dot-like or filamentous structures. Scale bars: 10 μm.

Induced Podocytes Possess Primary Processes with the Slit Diaphragm–Like Structures

We further analyzed the morphology of the induced glomeruli by electron microscopy. Both scanning and transmission electron microscopy showed well organized glomeruli surrounded by Bowman’s capsules (Figure 4, A and B). Interestingly, numerous microvilli were detected in the apical domain of the induced podocytes (Figure 4, C and D). Similar microvilli were reported in developing in vivo podocytes in humans.17,18 The podocytes were attached to each other at sites close to the basal region (Figure 4D). Inspection of the basal side of the induced podocytes by scanning microscopy identified multiple protrusions (Figure 4E), which were confirmed by transmission microscopy (Figure 4F). Higher magnification clearly showed bridging structures between the protrusions, which may represent an immature form of the slit diaphragm (Figure 4, G and H, Supplemental Figure 3, A–C). Thus, this is the first in vitrogeneration of mammalian podocytes with slit diaphragm–like structures from pluripotent stem cells. However, because typical interdigitation of the protrusions is lacking, they are likely to represent primary processes but not secondary processes (foot processes).

Figure 4.

Induced podocytes possess primary processes with the immature slit diaphragm–like structures. (A and B) Induced glomerulus covered with a Bowman’s capsule shown by (A) scanning and (B) transmission electron microscopy. (C) Induced podocytes observed by scanning electron microscopy. Multiple microvilli are observed on the apical surface (arrowheads). (D) Aligned podocytes, which attach to each other at sites close to the basal region, shown by transmission electron microscopy. Multiple microvilli are observed on the apical surface (arrowheads). (E) Primary processes shown by scanning electron microscopy (asterisks). Podocytes from the basal side are shown. (F) Primary processes shown by transmission electron microscopy (asterisks). (G) Slit diaphragm–like structures between the primary processes (arrows), shown by scanning electron microscopy. (H) Primary processes with slit diaphragm–like structures (arrows), shown by transmission electron microscopy. Scale bars: A and B: 10μm; C–F: 2 μm; G and H: 0.2 μm.

Induction of Podocytes from Human NPHS1-GFP iPS Cells Enables Their Efficient Isolation

We next tried to purify the GFP-positive podocytes at day 9 by FACS. Of the induced cells, 7.45%±0.72% (mean±SEM from five independent induction experiments) were positive for GFP (Figure 5A, left panel). We also found that the monoclonal antibody against the extracellular domain of nephrin (48E11),19in combination with the anti-podocalyxin antibody, was useful for sorting developing podocytes. Of the GFP-positive cells, 94.0% were positive for both nephrin and podocalyxin (Figure 5A, middle panel), while most of the GFP-negative cells (97.5%) were negative for both markers (Figure 5A, right panel). Thus, GFP faithfully mimics nephrin expression and podocytes were enriched in the GFP-positive population. Quantitative RT-PCR analysis of sorted cells confirmed the differential expression of several podocyte markers, such asNPHS2 (encoding podocin) and synaptopodin (Figure 5B). When the sorted GFP-positive cells were cultured for 3 days, the cells expressed WT1 in nuclei and podocalyxin on the cell surface (Figure 5C). Nephrin and GFP were detected on the cell surface membrane and in the cytoplasm, respectively, at day 7 of culture, although expression levels were lower than before the start of the culture. These results indicate that induction from NPHS1-GFP iPS cells enables efficient isolation of developing human podocytes.

Figure 5.

Induction of podocytes from human NPHS1-GFP iPS cells enables their efficient isolation. (A) FACS analysis of induced tissues at day 9. Almost 8% of cells are positive for GFP in this representative experiment (left panel). Nephrin and podocalyxin (PODXL) expression in the GFP-positive or -negative fraction (middle and right panel, respectively). (B) Quantitative RT-PCR analysis of GFP-positive and -negative fractions. Average and SEM from three independent experiments are shown. β-ACT, β-actin; SYNPO, synaptopodin. (C) Immunostaining of podocytes cultured for the indicated times after sorting GFP-positive cells. Scale bars: 5 μm. (D) Venn diagram of the transcription profiles of podocytes. Microarray data of GFP-positive podocytes are compared with those of human adult glomeruli and murine podocytes.

GFP-Positive–Induced Podocytes Show Transcriptional Profiles That Overlap with Those of Mouse and Human Podocytes In Vivo

To obtain comprehensive transcription profiles of the iPS cell–derived podocytes, we performed microarray analysis at day 9. We detected 2985 probes that were enriched in GFP-positive podocytes compared with GFP-negative cells. Of these, the top 300 genes were used for unbiased cluster analysis against microarray data from a wide variety of human tissues (Supplemental Figure 4, A and C).20 Genes enriched in the GFP-positive podocytes had variable tissue specificity. For example, NPHS2 was selectively expressed in the kidney or fetal kidney tissues. However, synaptopodin andFOXC2 were sorted into the ubiquitously expressing cluster. Dendrin was assigned to a cluster enriched in the neuronal tissues. These results suggest a single molecule is not sufficient to confirm the identity of podocytes. Therefore, we compared our gene list of GFP-positive human podocytes with the published microarray analyses of adult human glomeruli and adult podocytes from Mafb-GFP transgenic mice.11,21 Overall, 190 probes were overlapping among the three gene sets (Figure 5D, Supplemental Table 1, Table 1). These included typical slit diaphragm–related genes, such as NPHS1, NPHS2,CD2AP,22 chloride intracellular channel protein 5 (CLIC5),23 and dendrin,24,25and basolateral adhesion molecules such as claudin 5 and integrinα3.26,27Phospholipase ε1 and nonmuscle myosin heavy chain 9 (Myh9), causative genes for hereditary kidney diseases,2830 were also included. Transcription factors that have important roles in podocyte development, including WT1, MAFB, FOXD1, and TCF21, as well as vascular attractants such as VEGFA and semaphorin, were also expressed.1,2,31 Interestingly, when these selected overlapping genes were used for the cluster analysis against the microarray data from various organs described above, kidney and fetal kidney were segregated as separate clusters, suggesting the kidney-biased features of the overlapping gene set (Supplemental Figure 4B).

Table 1.

Genes common to iPS cell–derived podocytes in vitro, human glomeruli, and mouse podocytes in vivo

We also identified genes common to GFP-positive podocytes and adult human glomeruli (Figure 5D, Supplemental Table 2), and genes common to GFP-positive podocytes and mouse adult podocytes (Figure 5D, Supplemental Table 3). The former includes BMP7,32 while the latter includes NEPH1 (KIRREL),FOXC2, ROBO2, and EPHRIN-B1.7,3336 These results indicated that the typical transcriptional profiles are well conserved among our podocytes generated in vitro as well as mouse and human podocytes in vivo. In addition, extracellular matrix components characteristic of glomeruli at the capillary loop stage,lamininα5/β2/γ1 isoforms (corresponding to laminin 521) and type IV collagenα4/α5,37 were detected, the latter of which is the causative gene for Alport syndrome. These data indicate that the transition to these mature forms from immature laminin 111 and collagen α1/α2 has already occurred in vitro.

Taken together, our podocytes induced in vitro possessed the typical features of those in vivo, not only in morphology but also in transcription profiles, further supporting the authenticity of our human iPS cell induction protocol. In addition, genes exclusively expressed in the GFP-positive podocytes are worthy of further investigation because they may include genes specific to developing human podocytes, a possibility that has not been addressed to date (Figure 5D,Supplemental Table 4).

 

Transplanted iPS Cell–Derived Nephron Progenitors Form Vascularized Glomeruli

We next examined whether glomeruli generated from iPS cells integrated with the vascular endothelial cells. The iPS cell–derived nephron progenitor spheres were induced by spinal cord for 1 day in vitro to initiate tubulogenesis and were then transplanted beneath the kidney capsule of immunodeficient mice. We also cotransplanted mixed aggregates of human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) because these cells are useful for the generation of vascularized organ buds in vitro.38,39 When these aggregates were transplanted using a conventional method that we used for the transplantation of mouse ES cell–derived nephron progenitors,13 minimal nephron differentiation was observed at 10 days after transplantation (n=4) (Figure 6A). Because human iPS cell–derived aggregates were larger (approximately 1000 µm in diameter) than those from mouse ES cells (approximately 600 µm) and were instantly flattened upon transplantation (Supplemental Figure 5A), we hypothesized that mechanical tension of the capsule may have hampered nephron differentiation. Therefore, we inserted two agarose rods of 1100 µm diameter in a V-shaped position to release tension and secure a space for the transplanted aggregates (Figure 6B). We also soaked the rods with VEGF to enhance vasculogenesis.31 As a result, we observed immature glomerular formation at day 10 in the transplants, accompanied by blood vessels integrating into these glomeruli (n=5) (Figure 6, C and D). The vessels were preferentially clustered in the glomeruli among the grafted tissue (Figure 6D), suggesting that the iPS cell–derived podocytes possess the potential to attract vasculature. This is also consistent with microarray data showing VEGFA expression in our induced podocytes.

Figure 6.

Transplanted iPS cell–derived nephron progenitors form vascularized glomeruli. (A) Hematoxylin-eosin sections of tissues at 10 days after transplantation using a conventional method. Right panel: magnified image of the square in the right panel. kid, kidney of the host mouse. (B) Method for transplantation using solid agarose rods. Right panel: macroscopic view of transplanted tissue under the kidney capsule. Ag, agarose rods. (C) Hematoxylin-eosin sections of the transplanted tissue at day 10 in the presence of the rods. Right panel: magnified images of the square. (D) Vascularized glomeruli at day 10. Staining of WT1 and CD31. Right panel: magnified image of the square in the left panel. (E) Hematoxylin-eosin section of the transplanted tissue at day 20. Middle and right panel: magnified images of the squares in the panels on their left, respectively. *Stromal cells. kid, kidney of the host mouse. (F) Vascularized glomeruli formed upon transplantation at day 20. Left panel: magnified images of panel E. Right panel: magnified image of the square in the left panel. Note the enlarged Bowman’s space. (G) The endothelial cells are of mouse origin. Staining of WT1 (magenta) and MECA-32, a marker for mouse-specific endothelial cells (green). (H) Hematoxylin-eosin staining showing red blood cells in the induced glomeruli. (I) Hematoxylin-eosin staining showing the eosin-positive precipitates in the Bowman’s space. (J) Staining of nephrin (magenta) and CD31 (green). Right panel shows the basal localization of nephrin. Scale bars: A, C–F, I: 100 μm; B: 1 mm; G, H, J: 10 μm.

At day 20 after transplantation, we observed enlarged transplanted tissues beneath the capsule (Supplemental Figure 5B). Histologic examination revealed excessive growth of stromal cells of human origin, which were presumably derived from nonrenal tissues that were coinduced with nephron progenitors from iPS cells (n=4) (Figure 6E, Supplemental Figure 5C). Nonetheless, glomeruli were formed and the blood vessels were well integrated into the glomeruli (Figure 6, F and G). Moreover, 90% (135 of 150) of the glomeruli contained red blood cells (Figure 6H). Indeed, some of the glomeruli showed an enlarged Bowman’s space and contained eosin-positive precipitation (Figure 6I), which might imply a small amount of urine production. Interestingly, endothelial cells in the induced glomeruli were of mouse origin (Figure 6G,Supplemental Figure 5D). HUVEC-derived endothelial cells were not integrated into the iPS cell–derived glomeruli but were located separately from the sites of nephron formation (Supplemental Figure 5E). Therefore, HUVEC may not be competent to interact with human podocytes.

The anti-human specific podocalyxin antibody stained the apical domains of the iPS cell–derived podocytes, but not those of the host mouse podocytes (Supplemental Figure 5F). Nephrin protein in induced podocytes was localized at the basal side that faced the vascular endothelial cells (Figure 6J), suggesting the emergence of filtering apparatus. Electron microscopic analyses of two additional samples at day 20 showed that iPS cell–derived podocytes accumulated around, and were closely associated with, endothelial cells (Figure 7A). The induced podocytes developed numerous complex cell processes, as well as a linear basement membrane, at interfaces with endothelial cells (Figure 7B). The distances between the cell processes of some podocytes were enlarged, and slit diaphragm–like structures were formed between the processes located above the basement membrane (Figure 7C). Each of these diaphragms appeared as an electron-dense line (approximately 35 nm wide, 10 nm thick) bridging adjacent cell processes of the iPS cell–derived podocytes (Figure 7D). This feature was also observed in vivo and differed from the immature ladder-like structure that was seen between adjacent podocytes cultured exclusively in vitro without transplantation (Figure 4). Endothelial cells also produced basement membrane, but it was not fused to that of the podocytes in most cases, thus forming double-layered structures (Figure 7E). Interestingly, endothelial cells were fenestrated with residual diaphragm, a characteristic feature of embryonic glomerular endothelial cells (Figure 7F).40Furthermore, an electron-dense substance was detected in the Bowman’s space (Figure 7C), as in Figure 6I, implying the possible presence of filtration. Taken together, glomeruli generated from human iPS cells were vascularized and had many morphologic features present in glomeruli in vivo.

Figure 7.

iPS cell–derived glomeruli in the transplants exhibited many morphologic features of those in vivo. (A) Induced podocytes surrounding the vascular endothelial cells and extending many cell processes, shown by transmission electron microcopy. (B) Complex cell processes of podocytes formed between the cells and above the basement membrane. (C and D) Formation of slit diaphragm–like structures (arrows) between the cell processes of induced podocytes. Note the electron-dense substance in the Bowman’s capsule (asterisk). (E) Formation of double-layered basement membranes, each derived from endothelial cells (white arrowheads) and induced podocytes. (F) Fenestrated endothelial cells with diaphragms (black arrowheads). bm, basement membrane derived from induced podocytes; en, endothelial cells. Scale bars: A: 1 μm; B, E: 0.5 μm; C, D, F: 0.2 μm.

Discussion

We have inserted GFP into the NPHS1 locus of human iPS cells and successfully differentiated them toward three-dimensional glomeruli. The GFP-positive–induced podocytes possessed apicobasal polarity and were equipped with primary processes and slit diaphragm–like structures. Furthermore, sorted podocytes exhibited typical transcription profiles that overlap with those in vivo. These findings underscore the authenticity of our induction protocol.NPHS1 promoter–driven GFP expression is a good indicator of glomerulus formation. Several groups have reported the induction of kidney tissues in vitro,13,4143 and our iPS cell lines will be useful for assessing the induction efficiency of glomeruli by each protocol. In addition, we successfully sorted human podocytes using a combination of anti-nephrin and anti-podocalyxin antibodies. These reagents will make genetic GFP integration unnecessary for the purification of podocytes from patient-derived iPS cells, and possibly from more complex in vivo tissues.

It is surprising that well organized glomeruli are formed without the other two components of glomeruli: mesangial and vascular endothelial cells. These two cell types are not derived from nephron progenitors, as shown by cell lineage analysis in mice,8,44,45 and indeed we did not detect these lineages in the induced glomeruli (Supplemental Figure 3D). Thus, glomeruli can self-organize their structures solely from the podocytes derived from nephron progenitors, without any inductive signals from mesangial cells or the vasculature. However, further maturation will be required to reproduce hereditary glomerular diseases. We developed a new transplantation technique using agarose rods to secure a space against the tension evoked by kidney capsules. This technical improvement led to the successful generation, for the first time, of vascularized glomeruli derived from human iPS cells. The induced podocytes exhibited complex cell processes with slit diaphragm–like structures, and linear basement membrane that ran along that of the endothelial cells was formed. Furthermore, endothelial cells were fenestrated, which is a characteristic feature of glomerular endothelial cells. Most experiments used agarose rods soaked with VEGF to potentially accelerate vasculogenesis; however, the absence of VEGF in the rods also caused the formation of vascularized glomeruli (Supplemental Figure 5G). Thus, we can at least conclude that the human iPS cell–derived podocytes expressed sufficient attractants, including VEGF, to recruit endothelial cells.

It is noteworthy that the integrated endothelial cells were of mouse origin from the host animals but were not derived from HUVECs, although both vascular sources were initially located in proximity to the iPS cell–derived transplants. Therefore, human podocytes recruited mouse endothelial cells despite species differences, while HUVECs may not be competent to interact with human podocytes. Even when we performed transplantation without HUVECs or MSCs, we observed vascularized glomeruli, suggesting that paracrine effects of these cells may also be minimal (Supplemental Figure 5H). The presence of double-layered basement membrane might be caused by the incomplete fusion between those derived from human podocytes and mouse endothelial cells, as observed when mouse embryonic kidney was transplanted onto a quail chorioallantoic membrane.46 Therefore, the identification of optimal sources for human endothelial cells is necessary.

While it is difficult to estimate the gestational age on the basis of the morphology of the individual glomeruli,47,48 waiting for a longer period after transplantation may help further maturation of induced podocytes. However, we observed an excessive growth of stromal, presumably nonrenal, cells in the transplants. Thus, it will be essential to develop methods to purify nephron progenitors for transplantation. At the same time, it is necessary to induce genuine stromal cells because both interstitial cells and mesangial cells are derived from renal stromal progenitors.45 At present, we have no evidence that proper mesangial cells exist in our vascularized glomeruli. Ideally, human endothelial and mesangial cells that correspond to those in the developing kidney should be combined. Although further induction studies, as well as imaging techniques to visualize the slit diaphragm with a higher resolution,49are needed to achieve this goal, our results will accelerate the understanding of human podocyte biology both in developmental and diseased states.

 

 

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Artificial Pancreas

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Artificial Pancreas Therapy Performs Well in Pilot Study

Fri, 11/20/2015 – by Wiley

http://www.mdtmag.com/news/2015/11/artificial-pancreas-therapy-performs-well-pilot-study

 

Researchers are reporting a breakthrough toward developing an artificial pancreas as a treatment for diabetes and other conditions by combining mechanical artificial pancreas technology with transplantation of islet cells, which produce insulin.

In a study of 14 patients with pancreatitis who underwent standard surgery and auto-islet transplantation treatments, a closed-loop insulin pump, which relies on a continuous cycle of feedback information related to blood measurements, was better than multiple daily insulin injections for maintaining normal blood glucose levels.

“Use of the mechanical artificial pancreas in patients after islet transplantation may help the transplanted cells to survive longer and produce more insulin for longer,” said Dr. Gregory Forlenza, lead author of the American Journal of Transplantation study. “It is our hope that combining these technologies will aid a wide spectrum of patients, including patients with diabetes, in the future.”

 

Artificial Pancreas Works for Length of Entire School Term

Daniel Walls, a 12-year-old with type 1 diabetes who has taken part in the trial.

An artificial pancreas given to children and adults with type 1 diabetes going about their daily lives has been proven to work for 12 weeks – meaning the technology, developed at the University of Cambridge, can now offer a whole school term of extra freedom for children with the condition.

Artificial pancreas trials for people at home, work and school have previously been limited to short periods of time. But a study, published today in the New England Journal of Medicine, saw the technology safely provide three whole months of use, bringing us closer to the day when the wearable, smartphone-like device can be made available to patients.

The lives of the 400,000 UK people with type 1 diabetes currently involves a relentless balancing act of controlling their blood glucose levels by finger-prick blood tests and taking insulin via injections or a pump. But the artificial pancreas sees tight blood glucose control achieved automatically.

This latest Cambridge study showed the artificial pancreas significantly improved control of blood glucose levels among participants – lessening their risk of hypoglycemia. Known as ‘having a hypo,’ hypoglycemia is a drop in blood glucose levels that can be highly dangerous and is what people with type 1 diabetes hate most.

Susan Walls is mother to Daniel Walls, a 12-year-old with type 1 diabetes who has taken part in the trial. She said: “Daniel goes back to school this month after the summer holidays – so it’s a perfect time to hear this wonderful news that the artificial pancreas is proving reliable, offering a whole school term of support.

“The artificial pancreas could change my son’s life, and the lives of so many others. Daniel has absolutely no hypoglycaemia awareness at night. His blood glucose levels could be very low and he wouldn’t wake up. The artificial pancreas could give me the peace of mind that I’ve been missing.”

“The data clearly demonstrate the benefits of the artificial pancreas when used over several months,” said Dr. Roman Hovorka, Director of Research at the University’s Metabolic Research Laboratories, who developed the artificial pancreas. “We have seen improved glucose control and reduced risk of unwanted low glucose levels.”

The Cambridge study is being funded by JDRF, the type 1 diabetes charity. Karen Addington, Chief Executive of JDRF, said: “JDRF launched its goal of perfecting the artificial pancreas in 2006. These results today show that we are thrillingly close to what will be a breakthrough in medical science.”

 

Highly Sensitive Biosensor Measures Glucose in Saliva

The glucose biosensor fabricated with flexible substrates can perform in a variety of curved and moving surfaces, including human skin, smart textile and medical bandage.

Diabetic patients have to monitor blood glucose regularly and frequently, but conventional method of taking blood sample for measuring glucose level is painful. It is therefore important to develop high performance biological sensors for monitoring the glucose level at a reasonable cost.

The challenge to develop biosensor to test glucose in saliva is that the amount of glucose in saliva is too small for detection, and it requires a super sensitive biosensor to perform the job. The biosensor developed by PolyU researchers is fabricated with a glucose oxidase enzyme (GOx) layer, which is sensitive to glucose alone and nothing else. By detecting the electrical current, the glucose level can be known. However, there can be interference with current from other possible biological elements in saliva, such as dopamine, uric acid and ascorbic acid. To block such interference, researchers have coated Polyaniline (PANI) / Nafion-graphene bilayer films between the top enzyme layer and gate electrode. The strong adhesion of this top layer to the GOx layer enables the latter to stabilize and perform well in glucose detection.

Our novel biosensor is selectively sensitive to glucose, accurate, flexible and low in cost. The highly sensitive biosensor shows a detection lower limit of 10-5 mmol/L, which is nearly 1000 times sensitive than the conventional device for measuring blood glucose. This means with this biosensor, as little as 5 gram of glucose in a standard swimming pool of 50 m x 25 m x 2 m can be detected. Between the wide range of glucose level from 10-5 mmol/L up to 10 mmol/L (equivalent to 5 g – 5000 Kg of glucose in a standard swimming pool), the biosensor demonstrates linear response, which is good enough for measuring the possible range of glucose in the human body. Accuracy of the biosensor has been ascertained through laboratory experiments with repeatable results using glucose solutions of known glucose levels.

The glucose biosensor fabricated with flexible substrates can perform in a variety of curved and moving surfaces, including human skin, smart textile and medical bandage. Thus, it has great potential for development into wearable electronic applications, such as wearable biosensor for analysis of glucose level in sweat during exercise. It can also be mass produced at a low cost of HK$ 3 to 5 per test chip, which is comparable or even cheaper than the currently available commercialized products. In addition, this newly invented transistor-based biosensor platform is highly versatile. By changing to suitable enzymes, the platform can be used to measure the level of uric acid and other materials in saliva. For instance, if the biosensor is fabricated with enzyme uricase (UOx) and Polyaniline (PANI) / Nafion-graphene bilayer films, the platform can specifically be sensitive to uric acid only and other interference signals can be blocked.

 

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Genomics, Proteomics and standards


Larry H. Bernstein, MD, FCAP, Curator

http://pharmaceuticalintelligence/7/6/2014/Genomics, Proteomics and standards

This article is a look at where the biomedical research sciences are in developing standards for development in the near term.

 

Let’s Not Wait for the FDA: Raising the Standards of Biomarker Development – A New Series

published by Theral Timpson on Tue, 07/01/2014 – 15:03

We talk a lot on this show about the potential of personalized medicine. Never before have we learned at such breakneck speed just how our bodies function. The pace of biological research staggers the mind and hints at a time when we will “crack the code” of the system that is homo sapiens, going from picking the low hanging fruit to a more rational approach. The high tech world has put at the fingertips of biologists just the tools to do it. There is plenty of compute, plenty of storage available to untangle, or decipher the human body. Yet still, we talk of potential.

Chat with anyone heavily involved in the life science industry–be it diagnostics or pharma– and you’ll quickly hear that we must have better biomarkers.

Next week we launch a series, Let’s Not Wait for the FDA: Raising the Standards of Biomarker Development, where we will pursue the “hotspots” that are haunting those in the field.

The National Biomarker Development Alliance (NBDA) is a non profit organization based at Arizona State University and led by the formidable Anna Barker, former deputy director of the NCI. The aim of the NBDA is to identify problem areas in biomarker development–from the biospecimen and sampling issues to experiment design to bioinformatics challenges–and raise the standards in each area. This series of interviews is based on their approach. We will purse each of these topics with a special guest.

The place to start is with samples. The majority of researchers who are working on biomarker assays don’t give much thought to the “story” of their samples. Yet the quality of their research will never exceed the quality of the samples with which they start–a very scary thought according toCarolyn Compton, a former pathologist, now professor of pathology at ASU and Johns Hopkins. Carolyn worked originally as a clinical pathologist and knows first hand the the issues around sample degradation. She left the clinic when she was recruited to the NCI with the mission of bringing more awareness to the issue of bio specimens. She joins us as our first guest in the series.

That Carolyn has straddled the world of the clinic and the world of research is key to her message. And it’s key to this series. As we see an increased push to “translate” research into clinical applications, we find that these two worlds do not work enough together.

Researchers spend a lot of time analyzing data and developing causal relationships from certain biological molecules to a disease. But how often do these researchers consider how the history of a sample might be altering their data?

“Garbage in, garbage out,” says Carolyn, who links low quality samples with the abysmal non-reproducable rate of most published research.

Two of our guests in the series have worked on the adaptive iSpy breast cancer trials. These are innovative clinical trials that have been designed to “adapt” to the specific biology of those in the trial. Using the latest advances in genetics, the iSPY trials aim to match experimental drugs with the molecular makeup of tumors most likely to respond to them. And the trials are testing multiple drugs at once.

Don Berry is known for bringing statistics to clinical trials. He designed the iSpy trials and joins us to explain how these new trials work and of the promise of the adaptive design.

Laura Esserman is the director of the breast cancer center at UCSC and has been heavily involved in the implementation of the iSpy trials. Esserman is concerned that “if we keep doing conventional clinical trials, people are going to give up on doing them.” An MBA as well as an MD, Esserman brings what she learned about innovation in the high-tech industry to treatment for breast cancer.

From there we turn to the topic of “systems biology” where we will chat with George Poste, a tour de force when it comes to considering all of the various aspects of biology. Anyone who has ever been present for one of George’s presentations has no doubt come away scratching your head wondering if we’ll ever really glimpse the whole system that is a human being. If there is one brain that has seen all the rooms and hallways of our complex system, it’s George Poste.

We’ll finish the series by interviewing David Haussler from UCSC of Genome Browser fame. Recently Haussler has worked extensively on an NCI project, The Cancer Genome Atlas, to bring together data sets and connect cancer researchers around the world. What is the promise and pitfalls David sees with the latest bioinformatics tools?

George Poste says that in the literature we have identified 150,000 biomarkers that have causal linkage to disease. Yet only 100 of these have been commercialized and are used in the clinic. Why is the number so low? We hope to come up with some answers in this series.

 

 

Why Hasn’t Clinical Genetics Taken Off? (part 2)

published by Sultan Meghji on Fri, 06/20/2014 – 14:49

 

In my previous post, I made the broad comment that education of the patient and front line doctors was the single largest barrier to entry for clinical genetics. Here I look at the steps in the scientific process and where the biggest opportunities lie:

The Sequencing (still)

PCR is a perfectly reasonable technology for sequencing in the research lab today, but the current configuration of technologies need to change. We need to move away from an expert level skill set and a complicated chemistry process in the lab to a disposable, consumer friendly set of technologies. I’m not convinced PCR is the right technology for that and would love to see nanopore be a serious contender, but lack of funding for a broad spectrum of both physics-only as well as physical-electrical startups have slowed the progress of these technologies. And waiting in the wings, other technologies are spinning up in research labs around the world. Price is no longer a serious problem in the space – reliable, repeatable, easy to use sequencing technologies are. The complexity of the current technology (both in terms of sample preparation and machine operation) is a big hurdle.

The Analysis (compute)

Over the last few years, quite a bit of commentary and effort has been put into making the case that the compute is a significant challenge (including more than a few comments by yours truly in that vein!). Today, it can be said with total confidence that compute is NOT a problem. Compute has been commoditized. Through excellent new software to advanced platforms and new hardware, it is a trivial exercise to do the analysis and costs tiny amounts of money ($<25 per sample on a cloud provider appears to be the going rate for a clinical exome in terms of platform & infrastructure cost). Integration with the sequencer and downstream medical middleware is the biggest opportunity.

The Analysis (value)

The bigger challenge on the analysis is the specific things being analyzed as mapped to the needs of the patient. We are still in a world where the vast majority of the sequencing work is being done in support of a specific patient with a specific disease. There isn’t even broad consensus yet in the scientific community about the basics of the pipeline (see my blog posthere for an attempt at capturing what I’m seeing in the market). A movement away from the recent trend in studying specific indications (esp. cancer) is called for. Broadening the sample population will allow us to pick simpler, clearer and easier pipelines which will then make them more adoptable. It would be a massive benefit to the world if the scientific, medical and regulatory communities would get together and start creating, in a crowdsourced manner, a small number of databases that are specifically useful to healthy people. Targeting things like nutrition, athletics, metabolism, and other normal aspects of daily life. A dataset that could, when any one person’s DNA is references, would find something useful. Including the regulators is key so that we can begin to move away from the old fashioned model of clearances that still permeate the industry.

The Regulators

Beyond the broader issues around education I referenced in my previous post, there is a massive upgrade in the regulation infrastructure that is needed. We still live in a world of fax machines, overnight shipping of paper documents and personal relationships all being more important than the quality of the science you as an innovator are bringing to bear.

Consider the recent massive growth in wearables, fitness trackers and other instrumentation local to the human body. Why must we treat clinical genetics simply as a diagnostic and not, as it should be, as a fundamental set of quantitative data about your body that you can leverage in a myriad of ways. Direct to consumer (DTC) genetics companies, most notably 23andme, have approached this problem poorly – instead of making it valuable to the average consumer, what they’ve done is attempted to straddle the line between medical and not. The Fitbit model has shown very clearly that lifestyle activities can be directly harnessed to build commercial value in scaling health related activities without becoming a regulatory issue. It’s time for genetics to do the same thing.

 

 

Development and Role of the Human Reference Sequence in Personal Genomics

Posted by @finchtalk on July 3, 2014

discovery in a digital world

 

 

 

A few weeks back, we published a review about the development and role of the human reference genome. A key point of the reference genome is that it is not a single sequence. Instead it is an assembly of consensus sequences that are designed to deal with variation in the human population and uncertainty in the data. The reference is a map and like a geographical maps evolves though increased understanding over time.

From the Wiley On Line site:

Abstract

Genome maps, like geographical maps, need to be interpreted carefully. Although maps are essential to exploration and navigation they cannot be completely accurate. Humans have been mapping the world for several millennia, but genomes have been mapped and explored for just a single century with the greatest advancements in making a sequence reference map of the human genome possible in the past 30 years. After the deoxyribonucleic acid (DNA) sequence of the human genome was completed in 2003, the reference sequence underwent several improvements and today provides the underlying comparative resource for a multitude genetic assays and biochemical measurements. However, the ability to simplify genetic analysis through a single comprehensive map remains an elusive goal.

Key Concepts:

  • Maps are incomplete and contain errors.
  • DNA sequence data are interpreted through biochemical experiments or comparisons to other DNA sequences.
  • A reference genome sequence is a map that provides the essential coordinate system for annotating the functional regions of the genome and comparing differences between individuals’ genomes.
  • The reference genome sequence is always product of understanding at a set point in time and continues to evolve.
  • DNA sequences evolve through duplication and mutation and, as a result, contain many repeated sequences of different sizes, which complicates data analysis.
  • DNA sequence variation happens on large and small scales with respect to the lengths of the DNA differences to include single base changes, insertions, deletions, duplications and rearrangements.
  • DNA sequences within the human population undergo continual change and vary highly between individuals.
  • The current reference genome sequence is a collection of sequences, an assembly, that include sequences assembled into chromosomes, sequences that are part of structurally complex regions that cannot be assembled, patches (fixes) that cannot be included in the primary sequence, and high variability sequences that are organised into alternate loci.
  • Genetic analysis is error prone and the data require validation because the methods for collecting DNA sequences create artifacts and the reference sequence used for comparative analyses is incomplete.

Keywords:DNA sequencing

 

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Richard Lifton, MD, PhD of Yale University & Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension

Curator: Aviva Lev-Ari, PhD, RN

 

Yale’s Lifton receives $3 million science prize at gala Silicon Valley ceremony

Friday, December 13, 2013

Bill Hathaway / 203-432-1322

Read this article on YaleNews

Richard Lifton, Sterling Professor of Genetics and chair of the Department of Genetics, has received a $3 million Breakthrough Prize in Life Sciences, created by top Silicon Valley entrepreneurs.

Lifton was one of eight scientists honored Dec. 12 with $21 million in prizes at gala ceremonies hosted by actor Kevin Spacey in Mountain View, California. Celebrities — including Conan O’Brien, Glenn Close, Rob Lowe, and Michael C. Hall — handed out awards to six winners of the life sciences prizes and two co-winners of the Breakthrough Prize in Fundamental Physics.

“Scientists should be celebrated as heroes, and we are honored to be part of today’s celebration,” said Google co-founder Sergey Brin and his wife, biologist and entrepreneur Anne Wojcicki, two of the event’s sponsors.

Lifton, who is also an investigator for the Howard Hughes Medical Institute, was recognized for his pioneering work to identify the genetic and biochemical underpinnings of hypertension, a disease that affects more than 1 billion people worldwide and that contributes to 17 million deaths annually from heart attack and stroke. Lifton and his colleagues identified patients around the world with exceptionally high or low blood pressure due to single gene mutations. They identified the mutated genes and established their role in salt reabsorption by the kidney and regulation of blood pressure. The work gave scientific rationale to limit dietary salt intake and suggested rational combinations of antihypertensive medications and development of new therapies.

Other sponsors of the event are Chinese internet entrepreneur Jack Ma and Cathy Zhang; Russian entrepreneur and venture capitalist Yuri Milner and his wife, Julia Milner; and Facebook founder Mark Zuckerberg and Priscilla Chan.

At the end of the ceremonies, which will be televised on the Science Channel at 9 p.m. on Jan. 27, Milner and Zuckerberg announced the creation of a $3 million Breakthrough Prize in Mathematics that will be awarded next year.

Additional information on the prizes can be found atwww.breakthroughprizeinlifesciences.org or www.fundamentalphysicsprize.org.


SOURCE

http://www.bizjournals.com/sanfrancisco/prnewswire/press_releases/California/2013/12/13/NY33121

THE DISCOVERY

Laliotis MD, Zhang J, Volkman HM, Kahle KT, Hoffmann, KE, Toka HR, Nelson-Williams C, Ellison, DH, Flavell, R, Booth, CJ, Lu Y, Geller, DS, Lifton, RP. Wnk4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule. Nature Genetics, in press

Earlier Research Results on this discovey
Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):680-4. Epub 2003 Jan 6.

Molecular pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4.

Wilson FH1Kahle KTSabath ELalioti MDRapson AKHoover RSHebert SCGamba GLifton RP.

Abstract

Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl(-) dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a “mirror image” of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCT-mediated Na(+) influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ((22)Na-influx reduced 50%, P < 1 x 10(-9), surface expression reduced 75%, P < 1 x 10(-14) in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.

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SOURCE

LISTEN TO AUDIO TAPE by Prof. Richard Lifton

http://streaming.yale.edu/opa/podcasts/audio/schools/health_and_medicine/lifton_092007.mp3

January 27, 2014
Richard Lifton

Yale’s Richard Lifton is one of eight world-changing researchers whose work is celebrated during a program airing tonight (Jan. 27) on the Science Channel at 9 p.m. EST.

Lifton, Sterling Professor of Genetics and chair of the Department of Genetics, received a $3 million Breakthrough Prize in Life Sciences, created by top Silicon Valley entrepreneurs.

The Science Channel program features the Dec. 12 ceremony where Lifton and others received their prize. The festivities were hosted by actor Kevin Spacey and featured such celebrities as Conan O’Brien, Glenn Close, Rob Lowe, and Michael C. Hall, as well as tech leaders Mark Zuckerberg, Larry Page, Sergey Brin, Anne Wojcicki, Jimmy Wales, and Yuri Milner.

SOURCE

http://news.yale.edu/2014/01/27/tonight-lifton-honored-star-studded-ceremony

Yale consortium awarded $6 million to study therapies for vascular disease

Tuesday, January 21, 2014


Contact

Helen Dodson / 203-436-3984

Stacey Buba / 203-432-1333

Read this article on YaleNews

An international research team spearheaded by William C. Sessa, the Alfred Gilman Professor of Pharmacology and professor of medicine (cardiology), has been awarded a $6 million Transatlantic Networks of Excellence grant from the Fondation Leducq in France.

Sessa will be the U.S. coordinator for the consortium as it explores the mechanisms of secreted microRNAs and microRNA-based therapies for vascular disease. Sessa will be joined by a European coordinator, Dr. Thomas Thum, director of the Institute for Molecular and Translational Therapeutic Strategies at Hanover Medical School in Germany, and five investigators including recent Yale recruit, Carlos Fenandez-Hernando, associate professor of comparative medicine. The grant will be distributed over five years.

Sessa is director of the vascular biology and therapeutics program and vice chairman of pharmacology at Yale School of Medicine.

Sessa has long worked at the intersection of pharmacology and cardiovascular disease. He is on the scientific advisory board of the William Harvey Research Institute and NIHR Biomedical Research Unit in London, and also served on the joint strategy committee for the Yale-UCL collaborative in cardiovascular research.

“I am grateful to Fondation Leducq for funding this new international collaboration to find new and effective ways to treat a disease that kills millions of people each year,” Sessa said. “We have assembled a fantastic team of world class scientists to tackle the basic questions of how microRNAs are packaged and transferred between cells, and their therapeutic potential in vascular diseases.”

Fondation Leducq is a French non-profit health research foundation. Its mission is to improve human health through international efforts to combat cardiovascular disease. To this end, Fondation Leducq created the Transatlantic Networks of Excellence in Cardiovascular Research Program, which is designed to promote collaborative research involving centers in North America and Europe in the areas of cardiovascular and neurovascular disease.

Yale has had two previous Leducq grants — to Dr. Richard Lifton, chair of genetics, and Dr. Michael Simons, director of the Yale Cardiovascular Research Center.

SOURCE

http://bbs.yale.edu/about/article.aspx?id=6569

International Activity

  • YALE-UCL Collaborative
    London, United Kingdom (2011)
    Dr. Lifton is on the Joint Strategy Committee for the Yale-UCL Collaborative, an alliance which will provide opportunities for high-level scientific research, clinical and educational collaboration across the institutions involved: Yale University, Yale School of Medicine, Yale-New Haven Hospital and UCL (University College London) and UCL Partners
  • Transatlantic Network on Hypertension-Renal Salt Handling in the Control of Blood Pressure
    France (2007)
    Drs Hebert and Lifton will join leading researchers in Switzerland, France and Mexico in a transatlantic collaboration aimed at pinpointing the kidney’s role in high blood pressure.

Education & Training

M.D.
Stanford University (1982)
Ph.D.
Stanford University (1986)

Honors & Recognition

  • National Academy of Sciences
  • The Basic Science Prize
    American Heart Association
  • Homer Smith Award
    American Society of Nephrology
  • MSD International Award
    International Society of Hypertension

Research Interests

Molecular genetics of common human diseases


Research Summary

The common human diseases that account for the vast majority of morbidity and mortality in human populations are known to have underlying inherited components. Advances in human genetics have made the identification of genetic variants contributing to these traits feasible. Such identification promises to revolutionize the diagnostic and therapeutic approaches to these disorders. We have focused on cardiovascular and renal disease. To date, we have identified mutations underlying more than 20 human diseases; these include a host of diseases that define molecular determinants of hypertension, stroke and heart attack. We have gone on from these starting points to use biochemistry and animal models to define the physiologic mechanisms linking genotype and phenotype. These findings have provided new insight into normal and disease biology, are identifying new pathways underlying disease pathogenesis, and are identifying new targets for development of novel therapeutics.

Extensive Research Description

Cardiovascular disease is the leading cause of death world-wide. Epidemiologic studies have identified hypertension, high cholesterol, diabetes and smoking as major risk factors. By investigation of rare families recruited from around the world that segregate single genes with large effect, we have identified genes that contribute to these traits, putting a molecular face on their pathogenesis. For example, we have identified mutations in 8 genes that cause high blood pressure (hypertension) and another 8 that cause low blood pressure. These mutations all converge on a final common pathway, the regulation of net salt reabsorption in the kidney. These findings have established the key role of variation in renal salt handling in blood pressure variation, and have led to changes in the approach to treatment of this disease in the general population. They have also identified new therapeutic targets that are predicted to have greater efficacy with reduced side effects. Finally, they have identified new signaling pathways involved in the regulation of blood pressure homeostasis. We have taken similar approaches to another common disease, osteoporosis, with the identification of gain of function mutations in LRP5, a component of the Wnt signaling pathway, in development of high bone density. This finding has led to intensive efforts to identify small molecules that impact this pathway to protect against and/or reverse osteoporosis in the general population. Ongoing studies use both emerging and novel approaches to identification of genes that contribute to disease burden in the population, and to understanding the pathways that link genes to disease. Mutations that affect blood pressure in humans. A diagram of a nephron, the filtering unit of the kidney, is shown. The molecular pathways mediating NaCl reabsorption in individual renal cells along the nephron are shown, along with the pathway of the renin-angiotensin system, a major regulator of renal salt reabsorption. Inherited diseases affecting these pathways are indicated, with hypertensive disorders in red and hypotensive disorders in blue. From Lifton, Gharavi, and Geller. Cell, 104:545-556, 2001.


Selected Publications

  • Mani, A., et al. (2007). LRP6 mutation in a family with early coronary disease and metabolic risk factors. Science 315:1278-82.
  • Ring, A.M., et al. (2007). An SGK1 site in WNK4 regulates Na+ channel and K+ channel activity and has implications for aldosterone signaling and K+ homeostasis. Proc. Natl. Acad. Sci. (USA) 104:4025-9.
  • Lalioti MD, Zhang J, Volkman HM, Kahle KT, Hoffmann, KE, Toka HR, Nelson-Williams C, Ellison, DH, Flavell, R, Booth, CJ, Lu Y, Geller, DS, Lifton, RP. Wnk4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule. Nature Genetics, in press.
  • Wilson FH, Hariri A, Farhi A, Zhao H, Peterson K, Toka HR, Nelson- Williams C, Raja KM, Kashgarian M, Shulman GI, Scheinman SJ, Lifton RP. A cluster of metabolic defects caused by mutation in a mitochondrial tRNA. Science, 306:1190-94, 2004.
  • Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, Wu D, Insogna K, Lifton RP. High bone density due to a mutation in LDL-receptor-related protein 5. New Engl J Med. 346:1513-1521, 2002.
  • Wilson FH, Disse-Nicodème S, Choate KA, Ishikawa K, Nelson-Williams C, Desitter I, Gunel M, Milford DV, Lipkin GW, Achard JM, Feely MP, Dussol B, Berland Y, Unwin RJ, Mayan H, Simon DB, Farfel Z, Jeunemaitre X, Lifton RP. Human Hypertension Caused by Mutations in WNK Kinases. Science, 293:1107-1112, 2001.
  • Lifton RP, Gharavi A, Geller DS. Molecular mechanisms of human hypertension. Cell, 104:545-556, 2001.
  • Geller DS, Farhi A, Pinkerton N, Fradley M, Moritz M, Spitzer A, Meinke G, Tsai TF, Sigler P, Lifton RP. Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy. Science, 289:119-123, 2000.
  • Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, Lifton RP. Paracellin-1, a renal tight junction protein required for paracellular Mg2+ reabsorption. Science, 285:103-106, 1999.

SOURCE
http://bbs.yale.edu/people/richard_lifton-3.profile

PubMed Results: 10

Select item 225138461.

Protein phosphatase 1 modulates the inhibitory effect of With-no-Lysine kinase 4 on ROMK channels.

Lin DH, Yue P, Rinehart J, Sun P, Wang Z, Lifton R, Wang WH.

Am J Physiol Renal Physiol. 2012 Jul 1;303(1):F110-9. doi: 10.1152/ajprenal.00676.2011. Epub 2012 Apr 18.

PMID:

 22513846

[PubMed – indexed for MEDLINE]

Free PMC Article

Related citations

Select item 165287062.

Haplotype analysis in the presence of informatively missing genotype data.

Liu N, Beerman I, Lifton R, Zhao H.

Genet Epidemiol. 2006 May;30(4):290-300.

PMID:

 16528706

[PubMed – indexed for MEDLINE]

Related citations

Select item 165282533.

Familial aggregation of primary glomerulonephritis in an Italian population isolate: Valtrompia study.

Izzi C, Sanna-Cherchi S, Prati E, Belleri R, Remedio A, Tardanico R, Foramitti M, Guerini S, Viola BF, Movilli E, Beerman I, Lifton R, Leone L, Gharavi A, Scolari F.

Kidney Int. 2006 Mar;69(6):1033-40.

PMID:

 16528253

[PubMed – indexed for MEDLINE]

Related citations

Select item 127823554.

Mice lacking the B1 subunit of H+ -ATPase have normal hearing.

Dou H, Finberg K, Cardell EL, Lifton R, Choo D.

Hear Res. 2003 Jun;180(1-2):76-84.

PMID:

 12782355

[PubMed – indexed for MEDLINE]

Related citations

Select item 113430495.

Glucocorticoid-remediable aldosteronism is associated with severe hypertension in early childhood.

Dluhy RG, Anderson B, Harlin B, Ingelfinger J, Lifton R.

J Pediatr. 2001 May;138(5):715-20.

PMID:

 11343049

[PubMed – indexed for MEDLINE]

Related citations

Select item 102327426.

Elevated ambulatory blood pressure in 20 subjects with Williams syndrome.

Broder K, Reinhardt E, Ahern J, Lifton R, Tamborlane W, Pober B.

Am J Med Genet. 1999 Apr 23;83(5):356-60.

PMID:

 10232742

[PubMed – indexed for MEDLINE]

Related citations

Select item 97986657.

Coincident acute myelogenous leukemia and ischemic heart disease: use of the cardioprotectant dexrazoxane during induction chemotherapy.

Woodlock TJ, Lifton R, DiSalle M.

Am J Hematol. 1998 Nov;59(3):246-8.

PMID:

 9798665

[PubMed – indexed for MEDLINE]

Related citations

Select item 95012578.

In vivo phosphorylation of the epithelial sodium channel.

Shimkets RA, Lifton R, Canessa CM.

Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):3301-5.

PMID:

 9501257

[PubMed – indexed for MEDLINE]

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Autotransplantation for relapsed or refractory non-Hodgkin’s lymphoma (NHL): long-term follow-up and analysis of prognostic factors.

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Investigational Bioengineered Blood Vessel: Humacyte Presents Interim First-in-Human Data at the American Heart Association (AHA) Scientific Sessions 2013

Reporter: Aviva Lev-Ari, PhD, RN

The investigational bioengineered blood vessels represent a research and development milestone in vascular tissue engineering, as this technology could have the potential to help reduce or avoid surgical interventions and hospitalizations for patients with end-stage renal disease.

The Humacyte investigational bioengineered blood vessels are manufactured in a novel bioreactor system. The investigational bioengineered vessels go through a process of decellularization, which is designed to render them potentially non-immunogenic and implantable into any patient. These investigational bioengineered vessels are designed to be stored off-the-shelf for up to 12 months under standard refrigerated conditions, including, if successfully developedand approved,  on-site in hospitals.

 

Gail Thornton
Media Relations, Humacyte
1 908 392 3420 MOBILE
gail@westmillconsulting.com

Jim Modica

West Mill Consulting

908-872-4919

Jim@westmillconsulting.com

Humacyte Presents Interim First-in-Human Data

For Investigational Bioengineered Blood Vessel at the American Heart Association (AHA) Scientific Sessions 2013

  • The Humacyte investigational bioengineered blood vessel technology represents a research and development milestone in vascular tissue engineering.
  • Interim data from 28 patients in a three-center, first-in-human study in Poland indicate that all of the investigational blood vessels to date remain open to blood flow (patent), with no indication of an immune response in recipients, no aneurysms, and flow rates and durability suitable for dialysis.
  • The interim data suggest that the Humacyte investigational technology may have the potential to have high patency rates.
  • Longer follow-up and additional clinical studies will be required to confirm these preliminary observations.

 

RESEARCH TRIANGLE PARK, N.C., November 20, 2013 –Humacyte, Inc., a pioneer in regenerative medicine, today announced the presentation of interim, first-in-human data from an ongoing, multi-center study in Poland, evaluating the company’s investigational bioengineered blood vessel in hemodialysis patients with End-Stage Renal Disease (ESRD). The data were presented by Dr. Jeffrey H. Lawson, M.D., Ph.D., at the American Heart Association Scientific Sessions 2013 in Dallas, Texas (abstract). Dr. Lawson is Professor of Surgery and Pathology with tenure at Duke University Medical Center (Durham, North Carolina, USA), and Director of the Vascular Research Laboratory and Director of Clinical Trials for the Department of Surgery. He is also Clinical Consultant to Humacyte.

This is the first time surgical data from patients have been reported for the Humacyte investigational bioengineered vessel; the interim data come from a cohort of 28 study participants out of a total of 30 that will ultimately be enrolled in the three-site study in Poland (http://clinicaltrials.gov/show/NCT01744418%20CLN-PRO-V001%20NCT01744418). The first patients were implanted with the investigational vessels in December, 2012, and the vessels were first used for hemodialysis in February, 2013. The primary endpoints of the study in Poland are safety, tolerability, and patency to be examined at each visit within the first six months after graft implantation. Patients will be followed for an additional six months.

The interim patient data suggest that the Humacyte investigational bioengineered vessel may potentially be associated with low rates of vessel clotting, low infection rates, and low rates of surgical interventions. Low rates of clotting and intervention are consistent with preclinical data from animal testing that indicated little intimal hyperplasia. Preclinical data also indicated that, in animals, investigational vessels were remodeled to become living and more similar to native tissue. To date in the Polish study, the investigational vessel has remained open to blood flow (patent), with no indication of an immune response in recipients, no aneurysms (abnormal widening or ballooning of part of an artery due to weakness in the blood vessel wall), and flow rates and durability suitable for dialysis. Longer follow-up and additional clinical studies will be required to confirm these preliminary observations.

Co-authors on the presentation were: Drs. Marek Iłżecki, Tomasz Jakimowicz, Alison Pilgrim, Stanisław Przywara, Jacek Szmidt, Jakub Turek, Wojciech Witkiewicz, Norbert Zapotoczny, Tomasz Zubilewicz, and Laura Niklason.

Described by Investigator as “Breakthrough Investigational Technology”

“Based on our experience to date, this is breakthrough investigational technology,” said Principal Investigator Prof. Tomasz Zubilewicz, M.D., Ph.D., head, Department of Vascular Surgery and Angiology, Medical University of Lublin, Poland. “The investigational bioengineered vessel seems like it could have the potential to be shown to be superior to synthetic grafts in vascular access for hemodialysis in all aspects. This technology also has potential for other areas of vascular surgery, including replacement of infected synthetic grafts.”

“We are very encouraged by the Humacyte investigational bioengineered vessel’s performance in end-stage renal disease patients,” said Dr. Lawson. “Tremendous medical need exists for vascular access grafts in patients with ESRD who require dialysis. Based on this interim data and other ongoing research, we believe that the investigational bioengineered vessel has potential to meet this significant need.”

Need to Overcome Limitations of PTFE Grafts

Currently available synthetic vessels made from polytetrafluoroethylene (PTFE) are subject to many complications and about half fail within a year, requiring replacement surgery. PTFE vessels tend to become blocked (have low patency rates), have high rates of stenosis (an abnormal narrowing in a blood vessel that can be associated with hemodialysis), and high intervention rates.

“We continue to make significant progress in our research and development program with the Humacyte investigational bioengineered blood vessel,” said Laura E. Niklason, M.D., Ph.D., professor and vice chair of Anesthesia, professor of Biomedical Engineering, Yale University, and founder, Humacyte. “With our current interim study data, all of the Humacyte vessels have remained open to blood flow, with 20 out of the 28 implants requiring no intervention to date. We are grateful to patients, investigators, regulators and the broader vascular community for their ongoing collaboration and support in advancing this science.”

Unmet Medical Need in Chronic Kidney Disease

The Humacyte investigational technology is being developed with the goal of pursuing approval for use in patients with chronic kidney disease, a major global health problem affecting 26 million Americans[i] and around 40 million people in the European Union (EU).[ii] Individuals who progress to end-stage renal disease (ESRD) require renal replacement therapy (hemodialysis or kidney transplant); more than 380,000 patients currently require hemodialysis in the U.S.[iii] and some 250,000 patients require hemodialysis or have had kidney transplants in the EU.[iv] The investigational bioengineered vessels, if successfully developed and approved for use in ESRD by regulatory authorities, could offer the potential for significant cost savings to the healthcare system. These investigational bioengineered vessels represent a research and development milestone in vascular tissue engineering, as this technology could have the potential to help reduce or avoid surgical interventions and hospitalizations for patients with ESRD.

Investigators Highlight Preliminary Experiences In Patients

The investigators involved with the study in Poland cited their clinical observations in connection with the release of the preliminary patient data obtained for the Humacyte investigational technology.

“It was an exciting experience to be involved with this study, and to participate in this potential breakthrough in vascular surgery. This investigational bioengineered vein is a promising development for vascular surgeons,” said Principal Investigator Prof. Jacek Szmidt, head of the Department of General, Vascular and Transplant Surgery, Medical University of Warsaw, Poland.

“The Humacyte investigational bioengineered vessel was very easy to handle during implantation in this study. The graft maintained excellent mechanical properties, and based on our team’s experience, the complication rate to date has been very low compared with synthetic grafts,” said Investigator Stanisław Przywara, M.D., Ph.D., senior assistant, Department of Vascular Surgery and Angiology, Medical University of Lublin, Poland.

“During implantation in this study, the Humacyte investigational vessel behaved very much like a native vein.  Anastomotic hemostasis was achieved almost immediately. Insertion of needles to perform hemodialysis was easy and as reported by our nephrologists, provides very good adequacy of hemodialysis,” said Investigator Marek Iłżecki, M.D., Ph.D., senior resident, Department of Vascular Surgery and Angiology, Medical University of Lublin, Poland.

U.S. Clinical Trial Started in May, 2013

A multi-center U.S. clinical trial began in May, 2013 under a U.S. Investigational New Drug (IND) application. The U.S. trial will involve up to 20 patients across three sites to assess safety and performance of the innovative, investigational bioengineered blood vessels to provide vascular access for hemodialysis in ESRD patients.

About the Investigational Bioengineered Blood Vessels

The Humacyte investigational bioengineered blood vessels are manufactured in a novel bioreactor system. The investigational bioengineered vessels go through a process of decellularization, which is designed to render them potentially non-immunogenic and implantable into any patient. These investigational bioengineered vessels are designed to be stored off-the-shelf for up to 12 months under standard refrigerated conditions, including, if successfully developed and approved,  on-site in hospitals. Subject to receipt of regulatory approval, these properties could make the investigational bioengineered vessels readily available to surgeons and patients, and could eliminate the wait for vessel production or shipping. Data from studies of the investigational bioengineered vessels in large animal models reflect resistance to thickening for up to one year, and the early human studies that are now underway will provide safety and performance data in patients to support a future application for regulatory approval.

About Humacyte

Humacyte, Inc., a privately held company founded in 2005, is a medical research, discovery and development company with clinical and pre-clinical stage investigational products. Humacyte is primarily focused on developing and commercializing a proprietary novel technology based on human tissue-based products for key applications in regenerative medicine and vascular surgery. The company uses its innovative, proprietary platform technology to engineer human, extracellular matrix-based tissues that are designed be shaped into tubes, sheets, or particulate conformations, with properties similar to native tissues. These are being developed for potential use in many specific applications, with the goal to significantly improve treatment outcomes for a variety of patients, including those with vascular disease and those requiring hemodialysis. The company’s proprietary technologies are designed to result in off-the-shelf products that, once approved, can be utilized in any patient. The company web site is www.humacyte.com.

Forward-Looking Statement

Information in this news release contains “forward-looking statements” about Humacyte. These statements, including statements regarding management’s projections relating to future results and operations, are based on, among other things, management’s views, assumptions and estimates, developed in good faith, all of which are subject to known and unknown factors that may cause actual results, performance or achievements, or industry results, to differ materially from those expressed or implied by such forward-looking statements.

 

References


[iv]http://www.ekha.eu/usr_img/info/factsheet.pdf

SOURCE

From: Gail Thornton <gail@westmillconsulting.com>
Reply-To: Gail Thornton <gail@westmillconsulting.com>
Date: Wed, 20 Nov 2013 09:24:32 -0800 (PST)
To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>
Subject: Re: American Heart Association: Humacyte Investigational Bioengineered Blood Vessels

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Humacyte, Inc., a pioneer in regenerative medicine, presented the results of foundational U.S. preclinical studies of its investigational bioengineered blood vessel at the American Society of Nephrology’s ‘Kidney Week 2013’ Annual Meeting in Atlanta, GA.

Reporter: Aviva Lev-Ari, PhD, RN

HUMACYTE

Media Contacts:

Gail Thornton

West Mill Consulting

908-392-3420

Gail@westmillconsulting.com

Jim Modica

West Mill Consulting

908-872-4919

Jim@westmillconsulting.com

Humacyte Highlights Preclinical Data

Of Its Investigational Bioengineered Blood Vessel

 

  • Humacyte investigational bioengineered blood vessel technology represents a research and development milestone in the field of vascular tissue engineering.
  • Preclinical data on the investigational bioengineered blood vessel were presented at the American Society for Nephrology ‘Kidney Week’ meeting.
  • The pre-clinical data suggest that the Humacyte technology may have the potential to be associated with lowered vessel clotting and incorporation with animal model tissues.

RESEARCH TRIANGLE PARK, N.C., November 13, 2013Humacyte, Inc., a pioneer in regenerative medicine, presented the results of foundational U.S. preclinical studies of its investigational bioengineered blood vessel at the American Society of Nephrology’s ‘Kidney Week 2013’ Annual Meeting in Atlanta, GA.

The scientific presentation – by Shannon L. M. Dahl, Ph.D., co-founder and vice president, Technology and Pipeline Development, Humacyte – summarized U.S. preclinical data of the company’s investigational bioengineered vessel technology, which is being developed for use as the first off-the-shelf, human-derived, artificial blood vessel. The presentation’s title was ‘Preclinical Dataset Supports Initiation of Clinical Studies for Bioengineered Vascular Access Grafts.’ Co-authors were: Jeffrey H. Lawson, M.D., Ph.D.; Heather L. Prichard, Ph.D.; Roberto J. Manson, M.D.; William E.Tente, M.S.; Alan P. Kypson, M.D.; Juliana L. Blum, Ph.D.; and Laura E. Niklason, M.D., Ph.D.

Potential Of Investigational Bioengineered Vessels Explored In Pre-Clinical Studies

These U.S. preclinical data suggest that the investigational bioengineered vessel may be associated with lowered vessel clotting and incorporation with animal model tissues. This investigational technology is being developed with the goal of pursuing approval for use in patients with chronic kidney disease, a major global health problem affecting 26 million Americans[i] and around 40 million people in the European Union (EU).[ii] Individuals who progress to end-stage renal disease (ESRD) require renal replacement therapy (hemodialysis or kidney transplant); more than 380,000 patients currently require hemodialysis in the U.S.,[iii] and some 250,000 patients require hemodialysis or have had kidney transplants in the EU.[iv]

In ESRD patients, synthetic vascular grafts are prone to wall thickening, which results in graft clotting. Such clotting is the major cause of graft failures. As a result, ESRD patients experience frequent hospitalization and re-operation. The investigational bioengineered vessels, if successfully developed and approved by regulatory authorities, could offer the potential for significant cost savings to the healthcare system if approved for use in patients who require vascular access for ESRD. These investigational bioengineered vessels represent a research and development milestone in the field of vascular tissue engineering, as this technology could have the potential to help reduce or avoid surgical interventions and hospitalizations for patients with ESRD.

First Off-the-Shelf Investigational Bioengineered Vessel In Clinical Studies

“In the preclinical studies described, our investigational bioengineered vessels were repopulated with cells and remodeled like living tissue in the animal model,” said Dr. Dahl. “These investigational bioengineered vessels are produced using donated human vascular cells and then go through a process that is intended to decellularize the investigational vessels to remove the donor identity from the newly created vessels. This process is designed to produce investigational human grafts with the potential to be implanted into any patient at the time of medical need, enabling our investigational product to become the first truly off-the-shelf engineered graft to have moved into clinical evaluation. Demonstrating safety and performance in patients with end-stage renal disease could set the stage for follow-on development of our technology in other vascular procedures, such as replacement or bypass of diseased vessels, of vessels damaged by trauma, or for other vascular procedures.”

In 2012, Humacyte submitted an Investigational New Drug (IND) application to the U.S. Food and Drug Administration to conduct a multi-center U.S. clinical trial, involving up to 20 patients across three sites. In this trial, which will assess safety and performance of the investigational bioengineered vessels to provide vascular access for hemodialysis in ESRD patients, the first investigational bioengineered vessel was implanted in the arm of a kidney dialysis patient at Duke University Hospital in June, 2013.

European studies are already underway; as part of a multi-center study in Poland, the first patients were implanted with the investigational vessels in December 2012 and the vessels were first used for hemodialysis in February 2013. The primary endpoints of the study in Poland are safety, tolerability, and patency, to be examined at each visit within the first six months after graft implantation (see clinicaltrials.gov).

Studies Planned in Additional Patient Populations

Humacyte also will carry out a study in Poland to test safety and performance of the investigational bioengineered vessel as an above-knee bypass graft in patients with peripheral arterial disease (PAD). The study began in October of this year.

First-in-human interim study results for the investigational bioengineered vessel technology from Humacyte will be presented on Wednesday, November 20, 2013, at the American Heart Association Scientific Sessions (abstract) in Dallas, TX.

About Investigational Bioengineered Blood Vessels

The Humacyte investigational bioengineered blood vessels are manufactured in a novel bioreactor system. The investigational bioengineered vessels go through a process of decellularization, which is designed to render vessels potentially non-immunogenic and implantable into any patient. These investigational bioengineered vessels are designed to be stored for up to 12 months under standard refrigerated conditions, including, if successfully developed and approved, on-site in hospitals. Subject to receipt of regulatory approval, these properties could make the investigational bioengineered vessels readily available to surgeons and patients, and could eliminate the wait for vessel production or shipping. Data from studies of the investigational bioengineered vessels in large animal models reflect resistance to thickening for up to one year, and the early human studies that are now underway will provide safety and performance  data in patients to support a future application for regulatory approval.

About Humacyte

Humacyte, Inc., a privately held company founded in 2005, is a medical research, discovery and development company with clinical and pre-clinical stage investigational products. Humacyte is primarily focused on developing and commercializing a proprietary novel technology based on human tissue-based products for key applications in regenerative medicine and vascular surgery.  The company uses its innovative and proprietary platform technology to engineer human, extracellular matrix-based tissues that are designed be shaped into tubes, sheets, or particulate conformations, with properties similar to native tissues. These are being developed for potential use in many specific applications, with the goal to significantly improve treatment outcomes for a variety of patients, including those with vascular disease and those requiring hemodialysis. The company’s proprietary technologies are designed to result in off-the-shelf products that, once approved, can be utilized in any patient. The company web site is www.humacyte.com.

Forward-Looking Statement

Information in this news release contains “forward-looking statements” about Humacyte. These statements, including statements regarding management’s projections relating to future results and operations, are based on, among other things, management’s views, assumptions and estimates, developed in good faith, all of which are subject to known and unknown factors that may cause actual results, performance or achievements, or industry results, to differ materially from those expressed or implied by such forward-looking statements.

# # #


[iv]http://www.ekha.eu/usr_img/info/factsheet.pdf

SOURCE

From: Gail Thornton <gail@westmillconsulting.com>
Reply-To: Gail Thornton <gail@westmillconsulting.com>
Date: Wed, 13 Nov 2013 16:47:00 -0800 (PST)
To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>
Subject: American Society of Nephrology Kidney Week – Humacyte Press Release

 

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Renal Function Biomarker, β-trace protein (BTP) as a Novel Biomarker for Cardiac Risk Diagnosis in Patients with Atrial Fibrilation

Curator: Aviva Lev-Ari, PhD, RN

Original Research | November 2013

β-Trace Protein and Prognosis in Patients With Atrial Fibrillation Receiving Anticoagulation Treatment

Juan Antonio Vílchez, BSc Pharm, PhD; Vanessa Roldán, MD, PhD; Sergio Manzano-Fernández, MD, PhD; Hermógenes Fernández, MD; Francisco Avilés-Plaza, MD, PhD; Pedro Martínez-Hernández, BSc Pharm, PhD; Vicente Vicente, MD, PhD; Mariano Valdés, MD, PhD; Francisco Marín, MD, PhD; Gregory Y. H. Lip, MD

From the University of Birmingham Centre for Cardiovascular Sciences (Drs Apostolakis and Lip), City Hospital, Birmingham, England; and the Division of Cardiovascular Medicine (Drs Sullivan and Olshansky), University of Iowa Hospitals and Clinics, Iowa City, IA.

Correspondence to: Gregory Y. H. Lip, MD, University of Birmingham Centre for Cardiovascular Sciences, City Hospital, Dudley Rd, Birmingham, B18 7QH, England; e-mail: g.y.h.lip@bham.ac.uk

Abstract

Background:  Atrial fibrillation (AF) is associated with a high risk of mortality and morbidity and it commonly coexists with chronic kidney disease. A biomarker of renal function, β-trace protein (BTP), has been implicated in the progression of cardiovascular disease. The aim of our study was to evaluate the association of BTP with adverse cardiovascular events, bleeding, and mortality in patients with AF.

Methods:  In a consecutive cohort of patients with nonvalvular AF receiving anticoagulation treatment, plasma BTP was determined using an automated nephelometer BN ProSpec System (Siemens) and related to estimated glomerular filtration rate (eGFR). We recorded adverse cardiovascular events (stroke, acute coronary syndrome, and acute pulmonary edema), major bleeding, and mortality.

Results:  We included 1,279 patients (48.6% men), aged 76 years (IQR, 71-81 years), who were followed up for 996 days (IQR, 802-1,254 days). During the follow-up, there were 150 cardiovascular events (annual rate, 3.99%), 57 embolisms (annual rate, 1.54%), and 114 major bleeding events (annual rate, 3.04%), and 161 patients died (annual rate, 4.32%). BTP levels were inversely associated with eGFR (P < .001). High BTP concentrations were significantly associated with embolic events (hazard ratio [HR], 4.64 [1.98-10.86]; P < .001), composite adverse cardiovascular events (HR, 1.93 [1.31-2.85]; P = .001), and mortality (HR, 2.08 [1.49-2.90]; P < .001), even after adjusting for CHAD2DS2-VASc (congestive heart failure, hypertension, age ≥ 75 years [doubled], diabetes mellitus, stroke [doubled], vascular disease, age 65 to 74 years, sex category) score and renal function. High BTP was associated with major bleeding events (HR, 1.88 [1.18-3.00]; P = .008), even after adjusting for the HAS-BLED (hypertension, abnormal renal/liver function, stroke, bleeding history or redisposition, labile international normalized ratio, elderly [> 65 years], drugs/alcohol concomitantly) score.

Conclusions:  We suggest that BTP, a proposed renal damage biomarker, may be a novel predictor of adverse cardiovascular events, major bleeding, and mortality in patients with AF. BTP may help refine clinical risk stratification in these patients.

SOURCE

http://journal.publications.chestnet.org/article.aspx?articleid=1730537

Editorials | November 2013

Predicting the Quality of Anticoagulation During Warfarin Therapy:The Basis for an Individualized Approach

Giuseppe Boriani, MD, PhD

From the Institute of Cardiology, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna.

Correspondence to: Giuseppe Boriani, MD, PhD, Institute of Cardiology, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy; e-mail: giuseppe.boriani@unibo.it

Chest. 2013;144(5):1437-1438. doi:10.1378/chest.13-1285

In medicine, there is an emerging tendency toward individualized medicine, that is, an approach to medicine based on available evidence, but enriched by the awareness of the inherent limitations of any “one size fits all” approach. As a matter of fact, diseases show individual differences with regard to onset and course, and individuals show different responses to drugs and interventions, thus suggesting the rationale for an individualized approach to disease treatments, able to predict individual responses. The most sophisticated approach to individualization and tailoring of medicine is personalized medicine, a broad and rapidly advancing field of health care that is informed by each person’s unique clinical, genetic, genomic, and environmental information.1 Treatment with vitamin K antagonists (VKAs) has been one of the traditional settings for individualization of treatment. The concept of personalized medicine specifically applies to warfarin dosing, a setting where knowledge of the complex polymorphic variants in the gene encoding cytochrome 2C9 (CYP2C9) and of the genetic variants in the gene encoding vitamin K epoxide reductase complex 1 (VKORC 1) may help to predict the interindividual variability in warfarin pharmacokinetics and pharmacodynamics, as well as warfarin-associated events and costs.2 However, it is still uncertain and unproven whether management of warfarin dosing guided by pharmacogenetics may improve patient outcomes.3

Biomarker Can Predict Events in Afib Patients

Published: Nov 6, 2013 | Updated: Nov 7, 2013

By Todd Neale, Senior Staff Writer, MedPage Today
Reviewed by Zalman S. Agus, MD; Emeritus Professor, Perelman School of Medicine at the University of Pennsylvania and Dorothy Caputo, MA, BSN, RN, Nurse Planner

Beta-trace protein (BTP), a biomarker that has been associated with both kidney damage and an increased cardiovascular risk, may help identify high-risk atrial fibrillation patients, researchers found.

Among patients with atrial fibrillation who were on stable oral anticoagulant therapy, high plasma levels of the protein were associated with significantly elevated risks of embolic events, adverse cardiovascular events, death, and major bleeding, according to Gregory Lip, MD, of the University of Birmingham in England, and colleagues.

Also, adding information about BTP levels modestly improved the predictive ability of models that included two established risk scores — CHAD2DS2-VASc and HAS-BLED — as indicated by higher C-statistics, they reported in the Nov. 5 issue of CHEST.

“This raises the possibility that BTP may help refine the clinical risk stratification for thrombotic or hemorrhagic events and mortality in these patients,” they wrote.

BTP has been proposed has a marker of renal damage, and it has also been associated with inflammation, atherogenesis, angina, vasomotor reactivity, and hypertension. Previous studies have also identified a relationship between BTP and the progression of cardiovascular disease.

In the current study, Lip and colleagues explored whether BTP levels were related to outcomes in 1,279 patients with nonvalvular atrial fibrillation who were on stable oral anticoagulant therapy with an international normalized ratio (INR) of 2.0 to 3.0. Their average age was 76.

The median estimated glomerular filtration rate at baseline was 71.28 mL/min/1.73 m2; BTP levels and renal function were inversely related (P<0.001).

The BTP cut-offs with the best sensitivity and specificity for predicting each of the endpoints varied — 0.561 mg/L for adverse cardiovascular events, 0.556 mg/L for embolic events, 0.670 mg/L for mortality, and 0.573 mg/L for major bleeds.

During a median follow-up of 2.7 years, cardiovascular events occurred at a rate of 3.99% per year, embolisms at 1.54% per year, deaths at 4.32% per year, and major bleeds at 3.04% per year.

After adjustment for renal function and the CHAD2DS2-VASc risk score — which incorporates congestive heart failure, hypertension, age, diabetes, stroke, vascular disease, and sex — a BTP level above the cutoff was associated with increased risks of cardiovascular events (HR 1.93, 95% CI 1.31-2.85), embolic events (HR 4.64, 95% CI 1.98-10.86), and mortality (HR 2.08, 95% CI 1.49-2.90).

Also, after adjustment for the HAS-BLED risk score — which takes into account hypertension, abnormal renal and liver function, stroke, bleeding history or predisposition, labile INR, age over 65, and concomitant use of drugs and alcohol — a high BTP level was associated with a greater risk of major bleeding (HR 1.88, 95% CI 1.18-3.00).

“We suggest that BTP, a proposed renal damage biomarker, may be a novel predictor of adverse cardiovascular events, major bleeding, and mortality in patients with atrial fibrillation,” the authors wrote.

They acknowledged some limitations of the analysis, however, including possible selection bias because all of the patients were on stable oral anticoagulant therapy, the measurement of renal function and BTP levels at a single time point only, and the exclusion of patients with end-stage renal disease.

SOURCE

http://www.medpagetoday.com/Cardiology/Arrhythmias/42751

These are promising early results, but the data include plenty of limitations. As the article notes, the researchers themselves acknowledge that their work only looked at patients on a regular oral anticlotting drug at a certain point in time. Further research must include a broader class of patients to determine if BTP can be a reliable biomarker to help identify atrial fibrillation patients with an added risk of other health problems.

As hard as it might be to spot atrial fibrillation patients at risk of more problems, doctors struggle to definitively identify the condition in the first place and apply targeted treatments. The med tech industry, meanwhile, is trying to fill the gap. Topera, a 2013 Fierce 15 winner, recently won U.S. and EU approval for a 3-D device and mapping tool designed to better detect cardiac rhythm problems such as atrial fibrillation in order to enable more targeted and accurate treatment. In late August, St. Jude Medical ($STJ) snatched up Endosense, which makes a cutting-edge irrigated ablation catheter designed to treat atrial fibrillation, and rival companies are developing or promoting electrophysiology treatments and other devices for the condition.

 SOURCE

From: FierceBiomarkers <editors@fiercebiomarkers.com>
Reply-To: <editors@fiercebiomarkers.com>
Date: Wednesday, November 13, 2013 10:31 AM
To: AvivaLev-Ari@alum.berkeley.edu
Subject: | 11.13.13 | Investigators flag new biomarkers for atrial fib

Articles related to Diagnosis of Atrial Fibrilation published on this Open Access Online Scientific Journal include the following:

Genetic Analysis of Atrial Fibrillation, Larry H Bernstein, MD, FCAP  and Aviva-Lev Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/10/27/genetic-analysis-of-atrial-fibrillation/

Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmiasand Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Oxidized Calcium Calmodulin Kinase and Atrial Fibrillation, Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/10/26/oxidized-calcium-calmodulin-kinase-and-atrial-fibrillation/

Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis, Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/04/28/genetics-of-conduction-disease-atrioventricular-av-conduction-disease-block-gene-mutations-transcription-excitability-and-energy-homeostasis/

On Devices and On Algorithms: Prediction of Arrhythmia after Cardiac Surgery and ECG Prediction of an Onset of Paroxysmal Atrial Fibrillation, Justin D. Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

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