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/
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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).
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
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.
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).
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.
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,28–30 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-B1.7,33–36 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.
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.
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,13,41–43 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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