s2fieldsofstudy: Biology Medicine

text:
Derivation of Human Induced Pluripotent Stem Cells from Patients with Maturity Onset Diabetes of the Young*

Background: Human induced pluripotent stem cells (hiPSCs) can be harnessed for development of novel therapeutics for metabolic disorders. Results: Karyotypically normal hiPSCs were derived from patients with MODY1, MODY2, MODY3, MODY5, or MODY8. Conclusion: hiPSCs were successfully derived from a variety of MODY patients. Significance: MODY-hiPSCs can be used to explore the role of MODY genes in the development and function of pancreatic islet cells. Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human “stem cell cassette” containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1–60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes
is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human "stem cell cassette" containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1-60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.
Maturity onset diabetes of the young (MODY) 2 is an autosomal dominant monogenic diabetic disease typically affecting individuals before the age of 25 years. To date, 11 (1). Various disease-causing genetic variants have been identified for each of the MODY genes, all of which affect pancreas and beta cell development, ultimately resulting in beta cell dysfunction and diabetes.
The advent of induced pluripotency (2) has provided an opportunity to derive human induced pluripotent stem cells (hiPSCs) from MODY patients to model the disease in vitro. Several studies have reported that human somatic cells, commonly skin fibroblasts, can be reprogrammed into hiPSCs. A number of groups have reported the generation of hiPSCs from patients with mitochondrial diabetes, type 1 and type 2 diabetes mellitus (3,4). However, given the complexity of type 1 and type 2 diabetes mellitus, the use of hiPSCs from these patients requires comparison with multiple controls that are appropriate for the genetic and environmental context. 3 In the case of MODY-hiPSCs, however, the single mutation in each MODY gene would be primarily responsible for disease because the patients develop diabetes without an immune attack or invoking insulin resistance.
Here we demonstrate, for the first time, the ability to derive hiPSCs from patients with five different MODY conditions (MODY1, MODY2, MODY3, MODY5, and MODY8). The MODY-hiPSCs are morphologically, molecularly, and functionally indistinguishable from human embryonic stem cells (hESCs) and other reported hiPSCs, collectively known as human pluripotent stem cells (hPSCs). The availability of MODY-hiPSCs for in vitro disease modeling will serve the scientific community in two timely areas of research. First, the differentiation of MODY-hiPSCs along the pancreatic lineage presents a unique model to explore whether specific MODY genes and/or transcription factors play key roles during human pancreas development. This is especially significant considering that the efforts to derive functional mature beta cells from hPSCs have been hampered by a lack of complete understanding of human pancreas developmental biology, particularly of the late developmental stages (5). Second, because the mechanisms that underlie defects in islet function in MODY patients are still not fully understood, the MODY-hiPSCs will serve as an invaluable resource for investigating the direct role of the transcription factors and genes involved in the regulation of beta cell secretory function.

EXPERIMENTAL PROCEDURES
Patient Selection, Skin Biopsies, and Cell Culture-Skin biopsies were obtained after informed consent from healthy controls and MODY patients at Joslin Diabetes Center and Haukeland University Hospital. All studies using human material were reviewed and approved by the Institutional Review Boards and in accordance with the principles of the Declaration of Helsinki. Four-to six-mm skin biopsies were obtained from the anterior aspect of the upper forearm and cultured in a 6-well plate.
Sequencing of MODY Genes-Genomic DNA was harvested from patient skin fibroblasts and MODY-hiPSCs. MODY mutations were confirmed via PCR of known mutated regions of the MODY genes followed by Sanger sequencing.
Derivation and Characterization of hiPSCs-Derivation of some hiPSCs was performed in the Joslin iPS Core Facility. Further details regarding cell culture, primer sequences, generation of hiPSCs from patient skin fibroblasts (6), quantitative real time PCR, immunostaining, teratoma assay, and cytogenetic analysis can be found in the supplemental Experimental Procedures.

Characteristics of healthy individuals and MODY patients recruited at Joslin Diabetes Center and Haukeland University
Hospital following informed consent are included in Fig. 1, A and B. We devised a family node design of 2-4 closely related subjects (a duo, trio, or quad design) to obtain appropriate controls for the MODY patients (further explained in the supplemental Experimental Procedures). Fig. 1A includes examples of family node pedigrees for MODY1, MODY2, MODY3, MODY5, and MODY8 families. Genomic DNA was harvested from cultured skin fibroblasts and MODY gene mutations (HNF4A (p.Ile271fs), GCK (V62A), HNF1A (P291fsinsC), HNF1B (S148L and g.1-1671del), and CEL (C563fsX673)) were independently confirmed via PCR followed by Sanger sequencing (Fig. 2, A-E, and data not shown).
All hiPSCs derived in this study resemble hPSCs by morphologically presenting as distinct flat colonies (Fig. 2F), expressing pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1-60 (Fig. 2, G and H, supplemental Fig. S2, and data not shown), and exhibiting the ability to give rise to derivatives of the three germ layers (ectoderm, mesoderm, and definitive endoderm) in a teratoma assay (Fig. 2I, supplemental Fig. S1G, and data not shown). Interestingly, all the hiPSCs generated to date exhibit only one copy of viral integrant, allowing for easy excision when desired (supplemental Fig. S1H) (7). Normal karyotyping of MODY5-hiPSCs suggests that our method of reprogramming does not induce karyotypic abnormalities ( Fig.  2J and data not shown).

DISCUSSION
We have successfully derived hiPSCs from healthy individuals and patients diagnosed with MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), or MODY8 (CEL). These hiPSCs are morphologically, molecularly, and functionally indistinguishable from hPSCs, and representative karyotyping of MODY5-hiPSCs indicates that our reprogramming approach does not induce karyotypic abnormalities. The use of a polycistronic lentiviral vector for reprogramming facilitates high reprogramming efficiencies yet, unlike the commonly used set of four independent retroviruses, does not result in a large number of integration sites. Because our hiPSCs were generated with floxed alleles, the single copy of integrated "stem cell cassette" can be excised using Cre recombinase to obtain integrant-free hiPSCs and avoid nonspecific effects. Together, these data indicate that our hiPSCs are unique tools for in vitro comparative disease modeling of the various MODY disorders.
The MODY-hiPSCs that we have derived are unique tools for in vitro disease modeling to study the role of HNF4A, GCK, HNF1A, HNF1B, and CEL in human pancreas and beta cell development, as well as in beta cell dysfunction. Using MODY-hiPSCs from nondiabetic mutation carriers further offers the potential advantage of studying prediabetic cell biology. HNF4A (MODY1), HNF1A (MODY3), and HNF1B (MODY5) are members of the hepatocyte nuclear factor (HNF) family, implicated in pancreas development and function (8). HNF4A is important for the development and function of both beta cells and hepatocytes. Because MODY1 patients exhibit a diabetic phenotype that has been attributed to defective glucose-stimulated insulin secretion, the availability of MODY1-hiPSCs will allow in vitro modeling to determine the relevance of HNF4A mutations for liver development versus beta cell function. The transcriptional regulation of HNF1A by HNF4A (8) could partially account for the phenotypic similarities between the beta cells of MODY3 and MODY1 patients. As both MODY1 and MODY3 patients frequently exhibit diabetic microvascular complications, their hiPSCs can be used to derive endothelial cells to investigate mechanisms contributing to the complications. HNF1B has a critical role in both pancreas and kidney development. MODY5 patients are known to develop pancreatic hypoplasia and/or agenesis, and several patients manifest hypoplastic glomerulocystic kidney disease that is independent of the diabetes. Thus, MODY5-hiPSCs can be used to investigate the impact of mutations in HNF1B on both pancreas and kidney development.
GCK (MODY2) is the glucose sensor in the beta cell, and several inactivating mutations in GCK lead to impaired enzymatic activity and hyperglycemia (9). However, as GCK also processes glucose in hepatocytes, the individual impacts of the gene mutation on the two tissues and their synergistic role in the overall phenotype in MODY2 patients are not fully understood. CEL (MODY8), expressed in acinar cells, is a carboxyl ester lipase (10), and patients with CEL mutations develop pancreatic exocrine disease preceding diabetes. It is not known whether exocrine pancreatic dysfunction directly contributes to the diabetes phenotype in these patients. Thus, in both these scenarios, the differentiation of the respective MODY-hiPSCs along the pancreatic (endocrine and exocrine) lineage would potentially unmask mechanisms by which GCK and CEL mutations underlie defects in beta cell function and/or promote diabetes in humans.
In all the studies discussed above, the MODY-hiPSCs present a timely opportunity to pursue preclinical proof-ofconcept experiments. Correcting the mutations using transcription activator-like effector nucleases (11) would allow for the comparison of the pancreatic differentiation potential of these repaired MODY-hiPSCs with their autologous mutant counterparts. Subsequent studies utilizing the repaired isogenic controls and resulting in the reversion of in vitro derived phenotype would be strong proof of successful disease modeling. This approach is particularly appealing for MODY given the known genetic variants of each form.
In summary, we report the successful derivation of MODY-hiPSCs as a powerful resource for studying the role of these MODY genes/transcription factors in the development of human pancreas and beta cells and in the regulation of beta cell secretory function.


extfieldsofstudy: Biology Medicine

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MicroRNA-137 is downregulated in glioblastoma and inhibits the stemness of glioma stem cells by targeting RTVP-1

Glioblastomas (GBM), the most common and aggressive malignant astrocytic tumors, contain a small subpopulation of cancer stem cells (GSCs) that are implicated in therapeutic resistance and tumor recurrence. Here, we study the expression and function of miR-137, a putative suppressor miRNA, in GBM and GSCs. We found that the expression of miR-137 was significantly lower in GBM and GSCs compared to normal brains and neural stem cells (NSCs) and that the miR-137 promoter was hypermethylated in the GBM specimens. The expression of miR-137 was increased in differentiated NSCs and GSCs and overexpression of miR-137 promoted the neural differentiation of both cell types. Moreover, pre-miR-137 significantly decreased the self-renewal of GSCs and the stem cell markers Oct4, Nanog, Sox2 and Shh. We identified RTVP-1 as a novel target of miR-137 in GSCs; transfection of the cells with miR-137 decreased the expression of RTVP-1 and the luciferase activity of RTVP-1 3'-UTR reporter plasmid. Furthermore, overexpression of RTVP-1 plasmid lacking its 3'-UTR abrogated the inhibitory effect of miR-137 on the self-renewal of GSCs. Silencing of RTVP-1 decreased the self-renewal of GSCs and the expression of CXCR4 and overexpression of CXCR4 abrogated the inhibitory effect of RTVP-1 silencing on GSC self-renewal. These results demonstrate that miR-137 is downregulated in GBM probably due to promoter hypermethylation. miR-137 inhibits GSC self-renewal and promotes their differentiation by targeting RTVP-1 which downregulates CXCR4. Thus, miR-137 and RTVP-1 are attractive therapeutic targets for the eradication of GSCs and for the treatment of GBM.

IntroductIon
Glioblastoma (GBM), the most common and malignant primary brain tumors, are highly invasive, proliferative and vascularized [1]. The median survival of patients with GBM, following tumor resection and treatment with radiation and chemotherapy, is less then 16 months [1][2][3]. The dismal prognosis of these patients is mainly due to the infiltrative nature of GBM and the resistance of residual tumor cells to current therapeutic modalities [4,5].
GBM contain a small subpopulation of selfrenewing and tumorigenic cancer stem cells (glioma stem cells, GSCs) that are implicated in tumor infiltration, resistance to conventional therapies and tumor recurrence www.impactjournals.com/oncotarget [4,5]. Therefore, understanding the mechanisms associated with the stemness and oncogenic features of these cells is essential for the development of therapeutic approaches that can eradicate GSCs and may provide the basis for the development of novel therapeutic approaches to treat GBM patients [6].
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by either degradation or repression of mRNA translation [7,8]. miRNAs cause gene silencing by partial sequence homology and thus a single miRNA can have hundreds of targets and therefore regulate diverse cellular functions [8]. Indeed, alteration in miRNA expression has been associated with various disorders including cancer [9,10].
Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as an important mechanism of gene inactivation. CpG island hypermethylation has been described in almost every tumor type and many cellular pathways are inactivated by this type of epigenetic pathway [22]. Recent studies have shown that the downregulation of various miRNAs in some tumors is also mediated by CpG island hypermethylation [23]. miR-137 is a brain-enriched miRNA which has been shown recently to be downregulated in GBM and is considered a putative tumor suppressor miRNA (24). In glioma cell lines, miR-137 induces cell cycle arrest and suppresses migration and invasion. Moreover, miR-137 induces neuronal differentiation of mouse NSCs and in primary GBM culture [24,25].
Here, we examined the expression of miR-137 in glial tumors and GSCs and its role in self-renewal and differentiation of these cells. In addition, we identified RTVP-1 as a novel target of miR-137 and demonstrated its effects on the self-renewal of GSCs downstream of miR-137 and upstream of CXCR4.

results expression of mir-137 in GbM specimens and its methylation status
We first examined the expression of miR-137 in GBM compared to normal brain specimens and in GSCs compared to NSCs using real-time PCR. As presented in Figure 1A, the expression of miR-137 was significantly lower in the GBM specimens that were examined compared to normal brains. In addition, the expression of miR-137 was significantly lower in all the GSCs compared to human NSCs ( Figure 1B). miR-137 contains CpG islands in its promoter, therefore, hypermethylation can play a role in the downregulation of this miRNA expression in the tumor specimens. We analyzed a region of CpG islands in miR-137 promoter ( Figure 1C) and found that this region is hypermethylated in GBM compared to normal brain specimens ( Figure 1D). Therefore, changes in methylation may contribute to the downregulation of miR-137 in GBM.

role of mir-137 in the differentiation and selfrenewal of nscs and Gscs
Recent studies indicated that miR-137 plays a role in the neuronal differentiation of mouse NSCs and glioma cell lines (24). We further examined the role of miR-137 in the differentiation of human NSCs and the differentiation and self-renewal of GSCs. We first examined the expression of miR-137 in NSCs and GSCs that were grown as neurospheres or were induced to differentiate. NSCs were plated on laminin-coated plates and were differentiated by incubation with NM media. As presented in Figure 2A, differentiation of the human NSCs resulted in increased levels of the neuronal marker β3 tubulin and the astrocytic marker GFAP. The differentiated cells also exhibited a large increase in the expression of miR-137 as compared to control undifferentiated neurosphere cultures ( Figure 2C). The differentiation of the GSCs was performed under similar conditions and resulted in increased expression of the differentiation markers, GFAP and β3-tubulin ( Figure 2B). Similarly, the differentiation of these cells was also followed by upregulation of miR-137 albeit to a lower level than its expression in the differentiated NSCs ( Figure 2C).
We then examined the effects of miR-137 on the differentiation of NSCs using lentivirus vector expressing pre-miR-137. As presented in Figure 2D, the transduction of the NSCs resulted in high levels of miR-137 as determined by real-time PCR and in a significant increase in the neuronal differentiation of these cells ( Figure 2E) with some decrease in their astrocytic differentiation ( Figure 2F). We then examined the effect of miR-137 on the differentiation of GSCs using two GSCs, HF2355 and HF2414 that express low levels of this miRNA. The GSCs were transduced with a lentivirus vector expressing pre-miR-137-GFP or control pre-miR-GFP vectors. Transduction of these cells resulted in an increased level of miR-137 as determined by real-time PCR ( Figure 2D) and by increased GFP expression in the transduced HF2414 neurospheres as analyzed by fluorescence microscopy ( Figure 2G). Transduction of the GSCs with pre-miR-137 induced an increase in the neuronal differentiation of these cells ( Figure 2E) and some increase in the expression of GFAP ( Figure 2F).
The ability to form neurospheres is one of the hallmarks of GSC stemness and correlates with the tumor-initiating and tumorigenic potential of these cells. We therefore evaluated the effect of miR-137 on the neurosphere formation of the GSCs. We employed three different GSCs, HF2354, HF2355 and HF2414 that express low levels of this miRNA and transduced them with a lentivirus vectors expressing pre-miR-137 or control pre-miR. Transduction of the GSC neurospheres with lentivirus vector expressing pre-miR-137 significantly decreased the secondary neurosphere formation by 6.6fold, 3.3-fold, and 2.8-fold, respectively, compared with GSCs that were transduced with a lentivirus vector expressing a control pre-miR ( Figure 2H).
Additionally, we showed that miR-137 decreased the expression of the stemness markers, Nanog, Oct4, Sox2 and Shh in the transduced HF2355 GSCs ( Figure 2I) and in the HF2414 and HF2354 GSCs (data not shown).
Transduction of the GSCs with pre-miR-137 did not induce cell death (data not shown) and therefore the decreased neurosphere formation was not due to this process. Age adjusted t-test, P=0.001. Results are normalized relative to the levels of S12 mRNA and are presented relative to a reference sample (p<0.005 for all tumor as compared to non-tumor specimens). Expression of miR-137 was also determined for ten GSCs compared to two NSCs (B) *P < 0.001. Methylation of miR-137 was determined in twenty five GBM and seven normal brain specimens. An amplicon, starting 300 bp upstream of and ending 167 bp downstream of the pre-miR-137 start codon was used for the methylation analysis, covering 32 CpG sites. Circles represent CpG site (C). Methylation was determined by Sequenom mass spectrometry for each CpG position in the specific amplicon (D). *P < 0.01.

rtVP-1 is a novel target of mir-137
To delineate the mechanism by which miR-137 inhibited the self-renewal of the GSCs, we searched for miR-137 target genes using "miRwalk"(www.ma.uniheidelberg.de/apps/zmf/mirwalk/), a database of predicted and published microRNAs, and found several putative miR-137 target genes that may play a role in GSC selfrenewal. We focused on RTVP-1 since we recently demonstrated that the expression of this gene is correlated with the degree of malignancy of astrocytic tumors and that it is involved in the regulation of the growth, survival and invasion of glioma cells [36,37].
To confirm that RTVP-1 is a miR-137 target gene, we transduced the HF2354 GSCs with a lentivirus vector expressing pre-miR-137 and examined the effect of this treatment on the expression of RTVP-1. As presented in Figure 3A and 3B, transduction of the HF2354 cells with pre-miR-137 significantly decreased the expression of both RTVP-1 protein and mRNA, respectively compared to cells transduced with a control pre-miR.
We then examined if RTVP-1 is a direct target of miR-137. As presented in Figure 3C, miR-137 appears to have a strong target bias for the 3'-untranslated region (3'-UTR) of RTVP-1; having two predictive seed sequences in this region ( Figure 3C). Using a luciferase reporter plasmid in which the 3'-UTR of the RTVP-1 was cloned downstream of luciferase gene (pEZX-MT01:: RTVP-1-3'UTR), we demonstrated that miR-137 mimic significantly decreased the luciferase activity of this construct in U87 glioma cells by 4-fold as compared with a control miRNA ( Figure 3D).
In addition, real-time PCR analysis of the same GBM and normal brain specimens, that were analyzed for the expression of miR-137 ( Figure 1A), demonstrated an inverse pattern of RTVP-1 expression; upregulation in GBM compared to normal brains ( Figure 3E). Collectively, these findings strongly indicate that RTVP-1 is a direct target gene of miR-137 in GBM.

rtVP-1 mediates the inhibitory effect of mir-137 on the self-renewal of Gscs
We then examined the role of RTVP-1 in the selfrenewal ability of GSCs. We used two GSCs, HF2359 and HF2485, which express low level of endogenous RTVP-1 and transduced them with a lentivirus vector expressing RTVP-1 that increased the expression of the RTVP-1 protein in these cells ( Figure 4A). The transduction of both HF2359 and HF2485 GSCs with RTVP-1 significantly increased the self-renewal of these cells by 1.6-fold and 2-fold, respectively, compared to GSCs that were transduced with a control lentivirus vector ( Figure 4B). We then examined the effect of RTVP-1 silencing on the selfrenewal of the HF2355 and HF2414 GSCs that express high levels of this protein. Transduction of the GSCs with lentivirus vectors expressing shRNAs targeting RTVP-1 decreased the expression of this protein ( Figure 4C) and inhibited the self-renewal of the transduced GSCs by 2.9-fold and 3.7-fold, respectively, compared with GSCs that were transduced with a lentivirus vector expressing a control shRNA ( Figure 4D). After demonstrating that RTVP-1 promotes the selfrenewal ability of GSCs we examined its role in miR-137 effect. In these experiments, the HF2355 GSCs were transduced with a lentivirus vector expressing a RTVP-1 plasmid lacking the 3'-UTR together with a lentivirus vector expressing pre-miR-137 or with a control pre-miR vector. GSCs that were transduced with pre-miR-137 and control vector exhibited a 3-fold decrease in the number of neurospheres formed compared to the GSCs that were transduced with pre-miR-control. In contrast, transduction with lentivirus vector expressing RTVP-1 lacking the 3'-UTR, abrogated the inhibitory effect of miR-137 on the

cXcr4 is down-regulated in rtVP-1 silenced Gscs and mediates rtVP-1 effects
CXCR4 was recently reported to play a role in the self-renewal of GSCs by disrupting the Shh-GLI-Nanog network [17]. We therefore examined whether this pathway is associated with the effect of RTVP-1 on the self-renewal of GSCs. We found that silencing of RTVP-1 decreased the expression of CXCR4 in the HF2355 and HF2414 GSCs ( Figure 5A). To further examine the role of CXCR4 inhibition on GSC self-renewal, we employed the chemical CXCR4 antagonist, AMD3100. The GSCs were pre-treated with AMD3100 (50 nM) for 2 hr prior to the beginning of the assay and it was then added to the cultures every three days thereafter. As demonstrated in Figure 5B, AMD3100 significantly decreased the selfrenewal of the GSCs, as was recently reported [17]. We then examined the role of CXCR4 in RTVP-1 effects and found that overexpression of CXCR4 in RTVP-1 silenced HF2355 and HF2414 GSCs abrogated the inhibitory effect of this silencing on the self-renewal of the GSCs ( Figures  5C and 5D). Thus, the decreased expression of CXCR4 in the RTVP-1 silenced GSCs partially mediated the decreased self-renewal of these cells.
Collectively, these results demonstrate the importance of miR-137 in controlling the stemness of GSCs by targeting RTVP-1 which in-turn decreases the expression of CXCR4 and its downstream targets Shh and Nanog ( Figure 5E).

dIscussIon
GBM are the main primary central nervous system (CNS) tumors in humans, consisting of 70% of all malignant primary brain tumors. These tumors have poor prognosis due to treatment resistance and invasiveness of residual tumor cells and recurrence after resection, characteristics that are attributed to GSCs. In this study we explored the role of miR-137 in the stemness features and self-renewal of GSCs.
We found that miR-137 was downregulated in GBM compared to normal brain specimens and in GSCs compared to NSCs. Our results of decreased expression of miR-137 in GBM is in accordance with recent reports [26].
The downregulation of miRNAs in some tumors may be mediated by CpG island hypermethylation. We found that the promoter region of miR-137 was hypermethylated in GBM as compared with normal brain specimens, which may account, at least in part, for the downregulation of miR-137 in these tumors. Similarly, the downregulation of miR-137 in squamous cell carcinoma of the head and neck, gastric cancer, bladder cancer and colorectal cancer has been also attributed to hypermethylation of pre-miR-137 promoter [27][28][29]. miR-137 plays an essential role in controlling the fate of embryonic NSCs. Indeed, in mouse NSCs, miR-137 regulates the dynamics between NSC proliferation and differentiation during neural development by forming a feedback regulatory loop with TLX and LSD1 and by negatively regulating cell proliferation and accelerating the neural differentiation of embryonic NSCs [25]. Another study reported increasing levels of miR-137 in mouse NSCs that were differentiated into neurons and that miR-137 induced neuronal and reduced astrocytic differentiation in mouse NSCs and glioma cells [24].
We found that the expression of miR-137 was increased in differentiated NSCs and GSCs and that miR-137 increased the neuronal differentiation of NSCs and the neuronal and some glial differentiation of the GSCs. Moreover, miR-137 decreased the self-renewal ability of the GSCs and the expression of the stem cell associated proteins Nanog, Shh, Oct4 and Sox2. Self-renewal or secondary neurosphere formation is one of the major features of cancer stem cells and has been associated with the tumorigenic capacity of these cells, rapid tumor progression and with patient clinical outcome [30]. Collectively, our results show that miR-137 can reduce the stemness signature and characteristics of GSCs by decreasing self-renewal and inducing differentiation of GSCs.
miR-137 has been reported to act as a tumor suppressor miRNA in various types of tumors. In addition, several miR-137 targets have been recently identified in different tumors including COX-2 in GBM, CDK-6 in glioma cell lines and lung cancer, CtBP1 in melanoma cells, estrogen-related receptor alpha in breast cancer, Cdc42 in gastric cancer cells, colorectal cancer cells and lung cancer and c-Met, YB1, EZH2, and MITF in melanoma cells [24,26,[31][32][33][34][35]. In this study we identified RTVP-1 as a novel target gene of miR-137 by luciferase reporter assay and by demonstrating that miR-137 downregulated the mRNA and protein level of RTVP-1 in GSCs.
RTVP-1 is highly expressed in astrocytic tumors as compared to normal brains and its expression is dependent on tumor grade [36]. This protein is also involved in the regulation of the growth and survival of glioma cells by activating c-Jun-NH2-kinase and by increasing the expression of Bcl-2, rendering glioma cells more resistant to the apoptotic effect of tumor necrosis factor-related apoptosis-inducing ligand and serum deprivation [36]. RTVP-1 also plays a role in mediating the effect of SRF on glioma cell migration [37] and in the increased invasion of these cells by activating matrix metalloproteinase 2 [36].
Our finding that miR-137 targets RTVP-1 suggests that the downregulation of RTVP-1 by miR-137 mediates at least some of the tumor suppressor effects of miR-137 in GBM and specifically the inhibition of GSC selfrenewal. Indeed, we found that overexpression of RTVP-1 in GSCs increased their self-renewal, whereas silencing of this gene caused a significant inhibition. Moreover, we demonstrated that overexpression of RTVP-1 plasmid lacking its 3'-UTR abolished the inhibitory effect of miR-137 on the self-renewal of GSCs. Collectively, these results indicate that the ability of miR-137 to inhibit the stemness characteristics of GSCs was at least partly mediated by targeting of RTVP-1.
We found that silencing of RTVP-1 decreased the expression of CXCR4 which has been associated with cancer stem cell characteristics in various types of tumors [38,39]. Moreover, overexpression of CXCR4 was able to abrogate the inhibitory effect of RTVP-1 silencing on the self-renewal of the GSCs, suggesting that the decrease in CXCR4 expression mediates at least in part the inhibitory effect of RTVP-1 silencing. CXCR4 has been implicated in the control of Shh-Gli-Nanog network and the self-renewal and expression of embryonic stem cell (ESC)-associated genes in GSCs [17,40]. A recent study identified CXCR4 as a target of the miR-302-367 cluster [17]. Our current results emphasize the important role of this protein in controlling the self-renewal and GSC characteristics downstream of multiple pathways.
The core signaling pathways involved in cancer stem cell self-renewal include the WNT, Hedgehog, Notch, BMI, PTEN, BMP and TGF-β pathways [6]. Recent studies report that in GSCs some of these pathways also involve miRNAs that can inhibit the self-renewal ability. Such pathways include the targeting of the Bmi-1 by miR-128 and the miR-302-367 cluster that drastically affects self-renewal and infiltration properties through CXCR4 repression and consequent disruption of the Hedgehog-Nanog network [17,19]. Tumor suppressors that inhibit cancer cell proliferation, such as p53,p16INK4a and p19ARF, also inhibit stem cell self-renewal [41]. Here we identified a novel pathway that plays a role in the selfrenewal ability and differentiation of GSCs. This pathway is regulated by the brain-enriched miRNA, miR-137 through direct down-regulation of its target gene RTVP-1.
Recently, it was reported that differentiation of GSCs decreases their tumorigenic potential and renders them more susceptible to different therapies [42]. Thus, the ability of miR-137 to decrease self-renewal and promote differentiation of GSCs can be employed as a novel therapeutic approach in which the oncogenic potential of the GSCs is abolished with minimal damage to normal cells in the tumor microenvironment as compared with other current therapies in GBM [43].
In summary, we found that miR-137 is downregulated in glial tumors and in GSCs and that its promoter is hypermethylated in GBM specimens. In addition, we demonstrated that the inhibitory effect of miR-137 on GSC self-renewal was at least partly mediated by decreasing the expression of the novel miR-137 target, RTVP-1 which led to the downregulation of CXCR4 expression. These results implicate miR-137 and RTVP-1 as new therapeutic targets that regulate self-renewal and differentiation of GSCs.

human tissue specimens
Frozen human non-neoplastic brain tissue and human GBM specimens were obtained from the Department of Neurosurgery at Henry Ford Hospital. All human materials were used in accordance with the policies of the Henry Ford Health System Institutional Review Board at Henry Ford Hospital and the committee specifically approved this study. According to the IRB protocol, a written informed consent was obtained from all patients for the use of their tumor material.

Bisulfite treatment and methylation analysis
DNA was extracted according to the DNeasy kit, spin-column standard protocol (Qiagen, Maryland USA). Methylation analysis was performed on DNA extracted from paraffin blocks of eight GBM specimens and eight normal brains. Bisulfite treatment was performed on 1µg DNA using EZ DNA methylation Gold Kit (Zymo Research, Orange, CA), according to the Sequenom® protocol (Sequenom, San Diego, CA). Final elution of C-T converted DNA was with 50µl l H 2 O.
Amplicons and primers within the promoter area were designed using SequenomEpiDesigner (www. epidesigner.com). An amplicon, which covers 32 CpG sites, starting 300bp upstream and ending 167 bp downstream of the pre-miR-137 start codon was used for the methylation analysis,.
PCR amplification of amplicon was carried out with 50 ng of bisulfite-treated DNA in a total volume of 25 µl with GoTaq buffer, 200 µM dNTPs, 5 mM MgCl 2 , 10 pmol of reverse and forward primers, and 2 U of GoTaq polymerase. PCR conditions were 95°C for 10 min followed by 35 cycles of 94°C for 20 sec, 60°C for 30 sec and 72°C for 1 min, and later 3 min in 72°C. PCR product was treated with Shrimp Alkaline Phosphatase, and was subjected to in vitro transcription into RNA and T-specific cleavage by RNAse A, all according to the Mass CLEAVE protocol (Sequenom®, San Diego, CA). Samples were purified with 6mg Resin and robotically dispensed onto 384-well spectro CHIPS by Mass ARRAY Nano dispenser (Sequenom, San Diego, CA), for the Sequenom mass spectrometry detection. Results were analyzed by EpiTyper software.

Gsc cultures and differentiation
All human materials were used in accordance with the policies of the Institutional Review Board at Henry Ford Hospital. For the generation of GSCs, fresh GBM specimens were processed as previously described (44,45) and spheroids were maintained in neurosphere medium (DMEM-F12 1/1, glutamine 10 mM, HEPES buffer 10 mM and sodium bicarbonate 0.025%) supplemented with bFGF (20 ng/ml) and EGF (20 ng/ml). The GSCs were examined for the expression of CD44, Bmi-1, CD133, Musashi-1, Sox2 and nestin, self-renewal, expression of astrocytic, oligodendrocytic and neuronal markers upon plating on poly-D-ornithine in serum-containing medium and for their tumorigenic potential in nude rats.
For the differentiation studies, the NSCs and GSCs were disaggregated into small neurospheres and were plated on laminin coated plates in F12 medium supplemented with 2 % N12 (NM medium). For studying the effects of pre-miR-137, the cells were maintained in DMEM-F12 1/1 supplemented with 2% FCS and in the absence of growth factors.

transfection and transduction of Gscs
Transfection of GSCs with the miR-137 mimic (SMARTpool, Thermo Scientific, Lafayette, CO) was performed by siIMPORTER (Millipore, Billerica, MA) according to the manufacturer's recommendations. A lentiviral vector (System Biosciences, Mountain View, CA) expressing pre-miR-137-GFP and lentivirus vectors expressing RTVP-1 and control shRNAs or CXCR4 and RTVP-1 lacking the 3'-UTR were packaged and used to transducer the GSCs and NSCs according to the manufacturer's protocol. www.impactjournals.com/oncotarget luciferase assay Cells were first transfected with appropriate plasmids in 12-well plates, and then were harvested and lysed for luciferase assay 48 h after transfection. Luciferase assays were performed by using a luciferase assay kit (Promega, Madison, WI) according to the manufacturer's protocol. Renilla luciferase was used for normalization.

neurosphere formation assay
For analyzing the ability of GSCs to form secondary neurospheres, cells were plated in 24-well plates at a density of 100 cells/well through limiting dilution and the number of neurospheres/well was determined 14 days thereafter for eight different wells. Neurospheres that contained more than 20 cells were scored as previously described (44).

Western blot analysis:
Western blot analysis was performed as described. Equal loading was verified using an anti-β-actin or tubulin antibodies.

real-time quantitative Pcr analysis
Total RNA was isolated using TRIzol reagent (Invitrogen, Grand Island, NY) per the manufacturer's protocol and 1 μg of RNA was used to synthesize cDNA by SuperScriptase III (Invitrogen) with random primers. To detect the mRNAs of the different genes examined, the SYBR green method was used with the following primers: Shh -Forward; TCGGTGAAAGCAGAGAAC; reverse: AGGAAAGTGAGGAAGTCG; CXCR4 -forward: GTCATCTACACAGTCAACCTCTAC; reverse: ACCACCTTTTCAGCCAACAG; RTVP-1 -forward: GTGAAACCAACAGCCAGTGA; reverse TCTCTCCCAGTGAAGTGAAG; Oct4 -forward: ATCAGCCACATCGCCCAGCA; reverse: CCCAGCAGCCTCAAAATCCT; Sox2 forward: TGGGTTCGGTGGTCAAGTC; reverse: CGCTCTGGTAGTGCTGGGA and S12 Forward: TGCTGGAGGTGTAATGGACG, Reverse: CAAGCACACAAAGATGGGCT. Average level of S12 RNA was used as an internal control. Expression of miR-137 in cell lines or patient specimens was detected by the TaqMan stem-loop RT-PCR method. The primers and probes of the miR-137 U6 and RNU6B endogenous control for TaqMan miRNA assay were purchased from System Biosciences (Mountain View, CA).

statistical analysis
The results are presented as the mean values ± SE. The data of patient specimens are presented graphically with median and interquartile range noted. Data were analyzed using analysis of variance or a Student's t test with correction for data sets with unequal variances. Ageadjusted t-test is taken from a linear model including age as a covariate. Data were analyzed on a log 2 scale as appropriate.


text:
Identification of human nephron progenitors capable of generation of kidney structures and functional repair of chronic renal disease

Identification of tissue-specific renal stem/progenitor cells with nephrogenic potential is a critical step in developing cell-based therapies for renal disease. In the human kidney, stem/progenitor cells are induced into the nephrogenic pathway to form nephrons until the 34 week of gestation, and no equivalent cell types can be traced in the adult kidney. Human nephron progenitor cells (hNPCs) have yet to be isolated. Here we show that growth of human foetal kidneys in serum-free defined conditions and prospective isolation of NCAM1+ cells selects for nephron lineage that includes the SIX2-positive cap mesenchyme cells identifying a mitotically active population with in vitro clonogenic and stem/progenitor properties. After transplantation in the chick embryo, these cells—but not differentiated counterparts—efficiently formed various nephron tubule types. hNPCs engrafted and integrated in diseased murine kidneys and treatment of renal failure in the 5/6 nephrectomy kidney injury model had beneficial effects on renal function halting disease progression. These findings constitute the first definition of an intrinsic nephron precursor population, with major potential for cell-based therapeutic strategies and modelling of kidney disease.

INTRODUCTION
Nearly 26 million Americans, one in every nine, harbour kidney disease (Trivedi, 2010). Despite recent medical advances, the treatment options for patients with renal failure are limited. The alternatives available to patients who succumb to terminal renal disease are either supportive treatment in the form of dialysis or whole organ replacement by kidney transplantation. Dialysis is associated with long-term morbidity, mortality and poor quality of life. The shortage of donor organs and the long wait time on the recipient list hamper renal transplantation (Daar, 2006). The number of patients with terminal renal disease has increased, and the treatment costs for these patients now exceed the cumulative costs of treating cancer patients (Trivedi, 2010). Against this background of the growing number of patients with kidney disease and the limited treatment options, alternative treatments are clearly in need.
Various types of stem cells may be applicable as a platform for cell therapy for renal disease (Pleniceanu et al, 2010;Harari-Steinberg et al, 2011). Nevertheless, it seems that: (a) bone marrow and blood stem cells do not generate nephron cell types (Duffield et al, 2005;Krause & Cantley, 2005;Dekel et al, 2006b) and (b) the existence of an adult kidney epithelial stem cell with wide nephrogenic potential is still tenuous. Hence, isolation of tissue stem/progenitor cells from foetal kidneys is an attractive option for replenishment of nephron cells (Pleniceanu et al, 2010;Harari-Steinberg et al, 2011). The mammalian kidney is formed via reciprocally inductive interactions between two mesoderm precursor tissues, the metanephric mesenchyme (MM) and the ureteric bud (UB) (Pleniceanu et al, 2010). In response to UB signals, induced MM cells acquire an epithelial phenotype (mesenchymal to epithelial transition; MET) to generate committed nephron progenitor populations and sequentially form the pretubular aggregate, renal vesicle and C-and S-shaped bodies that expand to give rise to the mature nephron (Pleniceanu et al, 2010). Recent lineage tracing experiments of cell populations in transgenic mouse models have established that the transcription factor Six2 signifies a multipotent progenitor cell in the MM that condensates to form cap mesenchyme (CM) around the UB and is capable of selfrenewing and of differentiating towards different types of nephron epithelia (Boyle et al, 2008;Kobayashi et al, 2008).
Nevertheless, only a few studies have utilized human foetal kidney (hFK) as starting material for regenerative purposes, and most employ tissue transplantation rather than derivation of specific human cell types suitable for in vitro manipulation and cell therapy (Dekel et al, 1997(Dekel et al, , 2002(Dekel et al, , 2003Hammerman, 2000). Progenitor cell types in the MM have been previously isolated from embryonic mouse kidneys by means of a supply of the nephrogenic inducer, Wnt4 (Osafune et al, 2006), or by growing cells as nephrospheres, which, although robustly propagated in vitro, failed to display renal epithelial/nephrogenic capacity (Lusis et al, 2010).
With the intention of prospectively isolating progenitor populations from hFK by flow cytometry, we carried out global gene analysis (Dekel et al, 2006a) followed by protein expression studies (Metsuyanim et al, 2009) to pinpoint phenotypic surface markers that would replace intra-cellular renal transcription factors not relevant for sorting from human tissue, among them the neural cell adhesion molecule 1 (NCAM1). In the current study, by utilizing clonal analysis, defined serum-free culture conditions, and cell sorting with NCAM1, we were able, for the first time, to isolate a progenitor population from mid-gestation hFKs strongly committed to nephron formation and displaying beneficial effects in a mouse model of chronic renal failure.

RESULTS
NCAM1 þ cells are enriched for embryonic renal stem cell markers There has been considerable effort to identify cell surface markers to prospectively isolate stem cell populations from the kidney. We have previously put forward NCAM1 as a candidate marker for this (Dekel et al, 2006a;Metsuyanim et al, 2009). In fact, repeated verification by microarray analysis comparing human foetal and adult kidneys revealed NCAM1 as the most differentially expressed cell surface marker (>23-fold). Work in transgenic mouse models has identified Six2 in the CM as self-renewing nephron stem/progenitor cells (Kobayashi et al, 2008). To compare expression patterns in the developing kidney we co-stained organ cultures of embryonic mouse kidneys with antibodies for Ncam1, Six2 and Cdh1/E-cadherin ( Fig 1A). As previously described in hFK sections (Metsuyanim et al, 2009), Ncam1 in mouse organ culture was found expressed in the CM, as well as the earliest differentiated structures (renal vesicle, comma and S-shaped bodies). Six2 expression was solely found in the CM. Expression of Cdh1 as marker for epithelial cells was found to be almost mutually exclusive with Six2 expression, confirming the pre-MET expression of Six2, although occasionally we found co-expression of Six2 and Cdh1 as cells were going through the MET. It is not clear if these cells still express new Six2 protein, or whether they simply have not lost the existing protein yet. Therefore, Ncam1 expression was found in pre-and post-MET stages.
We queried a genomic data set from the GUDMAP database based on cell-type-specific gene expression profiles derived from a series of GFP-transgenic mouse lines and FACS separated lineages, each representing a specific developmental compartment regulated temporally and spatially (Harding et al, 2011). In the mixed kidney sample ST 1.0 dataset, Ncam1 was clearly and strikingly elevated in P0-P3 CM, corresponding to the time of post-natal nephrogenic burst observed in the mouse (Ncam1 is strongly expressed in P1 CM, data not shown). To identify other genes whose expression was co-ordinately regulated with that of Ncam1, we used Pearson correlation across these normalized gene expression profiles and identified a group of genes that exhibit essentially identical activation in P0-P3 CM and become somewhat inhibited at P4, consistent with the cessation of nephrogenic burst activity (Supporting Information Fig S1). The Ncam1 expression module, highly active in the peripartum nephrogenic burst phase CM, remarkably disclosed an overlap among neurogenic and nephrogenic programs and highlighted key players in the nephrogenic differentiation process (Supporting Information Fig S2). Interestingly, many of these genes are targeted by miRs-200b and 204, which regulate epithelial-mesenchymal transitions (Mongroo & Rustgi, 2010) and by the H3K27 repressive chromatin modification. Based on these results, the population of Ncam1 þ cells likely was heterogeneous, comprising a mix of stem and more committed progenitor cells.
We next determined whether hFK cultures would retain the NCAM1 þ cells. For clinical applications in cell therapy, it is essential that cells can be maintained and expanded under defined serum-free conditions. We therefore cultured hFK cells in serum-free medium (SFM; Kreso & O'Brien, 2008) at lowdensity. Culturing in SFM generated compact colonies of small round/cuboidal cells after 14 days in culture, whereas culturing in serum-containing medium (SCM) yielded dispersed fibroblastic/spindle-shaped cells (Fig 1B). At the early stage of the isolation procedure serum may promote the selection and expansion of stromal lineages and also dedifferentiation via A. Mouse embryonic kidney organ culture stained for Ncam1, Six2 and E-cad as indicated. An enlargement of the Ncam/Six2 signal is shown to emphasize the nuclear localization of Six2. White arrow illustrates the absence of Six2 signal in E-cad positive cells. An occurrence of Six2/E-cad positive cells is indicated with the asterisk. Images were obtained using Nikon A1R confocal microscope with and processed in ImageJ/Fiji software. B. Morphology of hFK cells cultured in SFM or SCM after 3 days (passage 0 Day 3-left panels) and towards confluence (14 days in SFM or 7 days in SCM-right panels). Distinct borders appear in SFM cultures (arrows) whereas cells with different morphology (arrows) are observed in SCM culture. Cells were observed using a Nikon Digital Sight camera attached to a Nikon Eclipse TS100 microscope. C. qRT-PCR analysis of gene expression in hFK cells cultured in SFM (three independent replicates). hPRT1 was used as endogenous control and SCM cells were used as the calibrator sample for RQ calculation (therefore ¼ 1). Data were analysed using SDS 3.2 software. D. Representative FACS analysis of NCAM1 expression in hFK cells cultured in SFM at passage1. Data is presented in a histogram graph showing NCAM1 staining in blue and the isotype controls staining (negative control) in red. E. Immunofluorescence staining of NCAM1 (red) in total hFK cells cultured in SFM. Nuclei stained with Dapi (blue). Images were obtained using Olympus DP72 camera attached to Olympus BX51 fluorescence microscope and processed via cellSens standard software.
Having established that hFK cells cultured in serum-free defined conditions skewed towards epithelial commitment but not uninduced mesenchyme or stromal lineage, we reasoned that derivation of NCAM1 þ cells from hFK stroma where expression is abundant (Metsuyanim et al, 2009) would be eliminated while NCAM1 expressing cells originating from the nephrogenic pathway retained. Accordingly, culture and growth of hFK cells in SFM yielded a smaller NCAM1 expressing population (range 5-20% in SFM; Fig 2D and E) compared to SCM (>30% in SCM as described previously; Metsuyanim et al, 2009).
Cultured NCAM1 þ cells display essential stem/progenitor properties We next determined whether the cultured NCAM1 þ cells harbour stem cell characteristics. hFK cells were therefore expanded in SFM and NCAM1-positive cells were collected via immunosorting (Supporting Information Fig S3D and E) and analysed for the expression of nephron stem/progenitor cell markers. We utilized at least three human foetal samples that carry inherent variance and may hamper statistical significance. In the NCAM1 þ cell population, we found significant elevation of the nephron progenitor markers compared to NCAM1 À cells (Fig 2A). Since NCAM1 is not detected in the UB epithelial lineage, markers such as PAX2 which strongly express in UB and derivatives were less enriched. Importantly, analysis of E-CAD levels showed significant reduction in NCAM1 þ cells, indicating that under SFM conditions these cells resemble earlier stages of induction towards mature nephrons than the NCAM1 À subpopulation. NCAM1 þ immunosorted cells expanded in SCM showed less enrichment in nephron progenitor gene expression (Supporting Information Fig S3F). Expression analysis of NCAM1 þ cells with oligonucleotide microarrays that allow a broader view of transcriptional changes (Supporting Information  Table S1) showed up-regulation of multiple genes that positively regulate cell cycle/cell division/cell growth, concomitantly, with a reduction in genes associated with terminal epithelial differentiation (Supporting Information Table S2). Analysis at the protein level supported gene expression; immunofluorescent labelling of VIM and E-CAD revealed lack of E-CAD and positive VIM staining in NCAM1 þ cells, whereas NCAM À cells showed wide spread E-CAD expression (Fig 2B and C). In addition, SIX2 immunostaining demonstrated enhanced expression in NCAM1 þ cells (Fig 2D-G, Supporting Information Fig S4) revealing the presence of earlier NCAM1 þ SIX2 þ characteristically displaying bright nuclei and small cytoplasm and more differentiated NCAM1 þ SIX2 À cell populations ( Fig 2F). Thus, we confirmed the existence of SIX2 þ and SIX2 À sub populations in NCAM1 þ cells from hFK samples observed in the mouse embryonic kidney cultures ( Fig 1A).
Clonogenic capacity of NCAM1-positive and -negative cells was analysed by limiting dilution. When cells were plated under defined SFM conditions, clonogenic activity of hFK cells was found almost exclusively within the NCAM1 þ fraction (Fig 2H and I). Self-renewal capacity of NCAM1 þ cells was determined; cells were able to expand from one cell with no supporting cells to a clone that continued to expand up to three to four passages postsorting maintaining a stable phenotype (Supporting Information Fig S5A). Hence, nephron precursors, although harbouring propensity for self-renewal, do not undergo endless self-renewing divisions under these culture conditions. To further improve expansion of NCAM1 þ cells in SFM, we examined the addition of niche factors that have been shown to support mice nephron progenitors in vitro (Dudley et al, 1999). Only BMP7 in the absence of fibronectin increased cell numbers/survival by 50% on average, but activating WNT signalling failed to show a beneficial effect (Supporting Information Fig S5B and C).
Having established that NCAM1 enriches for clonogenic and progenitor properties of hFK cells, we determined whether specific targeting of the NCAM1 þ fraction would deplete these properties. We therefore studied the effects of an antibody drug conjugate (ADC), which targets and depletes NCAM1 expressing cells (IMGN901; humanized NCAM1 monoclonal antibody covalently linked to the cytotoxic agent DM1; Lutz & Whiteman, 2009; Pode-Shakked et al, 2013) on clonogenicity/ self-renewal capacity of hFK cells. Calibration assays were performed to determine optimal ADC dosing, thus enabling the elimination of the largest possible NCAM1 þ cell fraction from hFK cells without harming other cells (Supporting Information Fig S5D and E). hFK primary cultures expanded in SFM were treated with 55 nM IMGN901, while control samples were given huN901 (NCAM1 antibody without the DM1 toxin) or left untreated. After 7 days of treatment, IMGN901-treated cultures were nearly abolished of clonogenic capacity and clone failed to propagate (Fig 2J), confirming that the stem/progenitor activity is contained in the NCAM1 þ sub-population.
In vitro cultured NCAM1 þ cells retain in vivo nephrogenic potential To test the in vivo differentiation/organogenic capacities of SFM cultured NCAM1 þ cells we performed grafting experiments on the chorio-allantoic membrane (CAM) of a chick embryo, a highly vascularized site supplying developmental cues Noiman et al, 2011). We have previously established this as a model to analyse the ability of human kidney cell types to generate renal structures; non-renal cell types such as bone marrow mesenchymal stem cells (MSCs) have consistently failed Noiman et al, 2011). No glomerular structures are apparent in CAM grafts, as their complex assembly is not likely to be supported in the CAM assay. In this assay, testing with different numbers of grafted human foetal cells showed that only high cell numbers of unselected hFK cells (!2.5 Â 10 6 ) could generate tubule-like structures (Supporting Information Fig 6A-E). When 1 Â 10 5 -5 Â 10 5 NCAM1 þ were grafted we found identifiable tubular structures in 4/6 NCAM1 þ cell grafts (an order of magnitude compared to unselected cells), while same numbers of NCAM1 À cells completely failed to generate similar structures (0/6 grafts; Fisher exact test, p ¼ 0.03; Fig 3A-C). The human origin of hFK tubular structures RT-PCR analysis of gene expression in NCAM1 cell fractions. GAPDH was used as endogenous control and NCAM1 À cell were used as the calibrator sample for RQ calculation (therefore ¼ 1). Data were analysed using SDS 3.2 software and presented as average RQ AE SDEV of three replicates. ÃÃÃ p < 0.001, Ã p < 0.05 versus NCAM1 À .
B-D. Immunofluorescence staining of NCAM1 þ and NCAM1 À subpopulations for E-cadherin (E-cad) (B, red) vimentin (C, green) and SIX2 (D, green). Nuclei stained with Dapi (blue). (B-C) Images were obtained using Olympus DP72 camera attached to Olympus BX51 fluorescence microscope and processed via cellSens standard software, bar represents 200 mm. (D) Images were obtained using Zeiss LSM 510 confocal microscope, bar represents 50 mm.

E.
Fluorescent quantification of SIX2 immunostaining as represented in (D).
Representative morphology of NCAM1 þ and NCAM1 À clones. Cells were observed using a Nikon Digital Sight camera attached to a Nikon Eclipse TS100 microscope. J.

Research Article
www.embomolmed.org Renal developmental progenitors B,C. H&E staining of paraffin-embedded sections of 5 Â 10 5 NCAM1 À (B) and 5 Â 10 5 NCAM1 þ (C) cells. Yellow arrows mark the tubule-like formations. The images were obtained using Scion colour digital camera attached to an Olympus BX51TF microscope. D-F. Segment-specific IHC staining of DBA (D), LTA (E) and THG (F) in paraffin-embedded sections of 5 Â 10 5 NCAM1 þ cell grafts. Yellow arrows mark the stained tubule-like structures while orange arrows mark the unstained structures. Images were obtained using a Scion Corporation grayscale digital camera attached to an Olympus BX51. G,H. Segment-specific IF staining of DBA (G) and LTA (H) in paraffin-embedded sections of 5 Â 10 5 NCAM1 þ cell grafts. Hoechst 33342 (blue) was used for nuclear staining. Images were obtained using an Olympus AX70 motorized microscope and spectral unmixing which allows distinction of background from specific staining, using a multispectral imaging system (NuanceFX camera and software). on the CAM was confirmed by non-cross reactive humanspecific Ki-67 antibodies, also indicating proliferative capacity of formed tubules (Supporting Information Fig S6F-H). Additionally, the epithelial marker panCK intensely stained the tubular structures but not individual cells yet to differentiate and organize (Supporting Information Fig S6I-K). We further analysed tubular structures derived from NCAM1 þ cells for the presence of mature nephron cell types using markers specific for proximal (LTA; lotus tetragonolobus lectin), loop of Henle (anti-THG; Tamm-Horsfall glycoprotein) and distal (DBA; dolichos biflorus agglutinin) tubules. Immunostaining with LTA, DBA and anti-THG revealed the presence of all three (Fig 3D-H), which were devoid of NCAM1 expression (data not shown). Altogether, CAM grafting which enables the NCAM1 þ progenitors to undergo MET into mature epithelia demonstrates their enriched and broad intrinsic differentiation capacity that NCAM À counterparts lack.

Research Article
In vitro cultured NCAM1 þ hNPCs improve the outcome of chronic renal injury The data presented so far convinced us that the SFM cultured NCAM1 þ cell population represents committed human nephron progenitors (hNPCs). To demonstrate the clinical importance of hNPCs, we carried out proof-of-principle experiments evaluating functional renal parameters in a mouse model of renal injury. De novo generation of mature renal cell types is likely to be relevant in chronic progressive kidney disease (e.g. chronic proteinuric states), in which nephron cells are progressively depleted. We therefore tested the therapeutic effects of hNPCs in the 5/6 nephrectomy (Nx) chronic progressive kidney injury model. We generated 5/6 Nx in 44 NOD/SCID mice and implemented a protocol of three consecutive injections of 5 Â 10 5 hNPCs directly into the renal parenchyma (every 3 weeks) and followed treated mice (n ¼ 20) for a 14-week period in comparison to untreated mice (n ¼ 24, saline injected; Supporting Information Fig S7A). For confirmation of cell engraftment hNPCs were labelled with CellTracker and analysed 2 weeks after cell injection using an in vivo imaging system ( Fig 4A) and laser scanning confocal microscopy (Supporting Information Fig S7B). These data, along with the gene expression of human-specific b2-microglobulin (hb2M) in the diseased murine kidney (Fig 4B, Supporting  Information Fig S7C), indicated robust engraftment of hNPCs. The prolonged 5/6 Nx experiment enabled 24 h urinary collection and repeated measurement of creatinine clearance (CrCl) as a measure of renal function. For each time point we analysed the change from baseline CrCl (1 week post 5/6 Nx) among individual mice. At 12 weeks, the proportion of mice with a reduction of more than 15% in CrCl including mortality events was significantly lower in the hNPC treated group (5/20 mice) compared to the untreated saline group (15/24 mice; Fisher exact test, p ¼ 0.01, Fig 4C). In addition, at 12 weeks but not in earlier time points, there was significant improvement in change from baseline in the absolute CrCl values with mean differences per minute of 0.034 AE 0.04 ml in the hNPC group versus À0.0137 AE 0.019 ml/min in the untreated group (t-test, p ¼ 0.01; Fig 4D). Thus, treatment with hNPCs halted disease progression and increased CrCl throughout the 12-week study period, a finding that is consistent with an improvement in kidney function.
To further provide possible mechanistic insight as to the effects of hNPCs via production of renal epithelium and cell replacement we immunostained consecutive sections injured kidneys with human-specific markers, HLA and panCK/MNF116 (Cohen et al, 2010;Eventov-Friedman et al, 2005 and Supporting Information Fig S7D showing non-cross reactivity and specificity for human kidney epithelia). Three patterns of engraftment were observed, including integration into an existing mouse tubule (Fig 4E, F and H), between tubules ( Fig 4I) and tubular regeneration (Fig 4E and G) consisting of small early tubules staining for HLA, panCK and lacking NCAM1 that appeared within 2 weeks ( Fig 4J) and larger mature-appearing epithelial tubules with a patent lumen after 3 months (Fig 4K). Furthermore, immunostaining with distal (EMA) and proximal (ENPEP) markers disclosed renal identity (Fig 4L and Supporting Information Fig S7E;Fig 4M and Supporting Information Fig S7F,respectively). Altogether, repeated injection demonstrated that hNPCs have integrated to a certain extent into the endogenous mouse tissue, mixed with pre-existing host cells, and regenerating injured kidney tissue.

DISCUSSION
Using defined culture conditions and NCAM1 immunosorting, we succeeded for the first time in prospectively isolating committed nephron progenitors from hFK. We showed that hNPCs have primitive characteristics that express distinct nephron progenitor markers. hNPCs also expressed relatively high levels of VIM and low levels of CDH1/E-CAD, corresponding to early stages of epithelial induction and confirming their primitive phenotype. Using clonal stem/progenitor cell assays, we found that NCAM1-expressing cells contain highly clonogenic and self-renewing cell populations (the latter to a limited extent in our culture conditions). Clonal hNPC were highly enriched for NCAM1 in SFM and NCAM1 targeting abrogated hFK cell clonogenicity. When grafted on the chick CAM these cells proved robustly tubulogenic in situ without the requirement of extra-stromal compartment and were more efficient at renal differentiation than were unsorted cells (whole hFK) and differentiated NCAM1 À counterparts. Importantly, foetal NCAM1 þ cells not only exhibited rapid initiation of tubulogenesis but also broad nephron epithelial potential.
Previously, because of its mesodermal derivation, the hFK and undifferentiated MM were used as a valuable source of CD90 þ plastic mesenchymal cells and MSC-like cells that gave rise to multiple lineages but not to kidney epithelial lineage (Almeida-Porada et al, 2002). Similarly, clonal progenitors isolated from the mouse embryonic kidney by means of selective growth of nephrospheres displayed broad mesodermal multipotentiality (MSC-like) but not epithelial capacity and are thought to be derived from undifferentiated MM (Lusis et al, 2010). We obtained similar results when single cells derived from hFK were seeded at low density in SCM and spindle MSC-like cells potentially originating from undifferentiated MM Research Article www.embomolmed.org Renal developmental progenitors or renal stroma emerged and became dominant in low passage. In fact, long-term self-renewing cell clones derived from our SCM cultures phenotypically lacked epithelial markers and resembled propagatable MSC populations commonly isolated by adhesion culture (Bruno et al, 2009;Dekel et al, 2006c). Importantly, in the chick CAM graft assay MSCs used as controls do not epithelialize to become nephron cell types . In contrast, when stimulated in vitro with serum-free defined conditions unsorted single hFK cells proliferated and readily acquired distinct morphology, inherent epithelial markers such as EpCAM and epithelial lineage commitment. NCAM1-expressing hNPCs, isolated from hFK cells expanded in SFM, are therefore clearly different both phenotypically and functionally from MSCs or MSC-like cells. At present it is not possible to precisely identify the developmental stage the hNPCs originate from. Although the SFM culture conditions enrich the cultures as a whole for epithelial markers, the NCAM1 þ sub-population appears to be less differentiated, as shown by their loss of CDH1/E-CAD and increase in SIX2 expression. During mouse kidney development expression of Cdh1/E-cad and Six2 was found to be almost mutually exclusive, with the exception of the cells that are going through the MET. One possibility is therefore that the hNPCs are this minor subset of E-CAD/SIX2 þ /NCAM1 þ cells. It is however not clear if these cells are truly expressing both SIX2 and E-CAD, or the observed co-expression is the result of SIX2 protein stability after its expression has been stopped. Alternatively, the hNPCs could be the complete SIX2 þ sub-population of the NCAM1 þ cells and therefore be derived from the CM. Further fractionation of the NCAM1 þ population in mouse and human may separate early (NCAM1 þ SIX2 þ ) and more committed nephron progenitors (NCAM1 þ SIX2 À ) and indicate functional potency. This was recently evident in Wilms' tumour for which an additional marker (ALDH) within the NCAM1 þ fraction was used to more precisely pinpoint cancer stem/initiating cells in the tumour (Pode-Shakked et al, 2013). We therefore, at this stage, collectively consider foetal human progenitors expressing NCAM1 as a pool of mitotically competent yet strongly nephrongenerating-biased cells.
Neither haematopoietic stem cells (HSCs) nor MSCs, the most accessible human stem cells, can be used to derive stem/ progenitor cells capable of generation of renal components (Almeida-Porada et al, 2002;Harari-Steinberg et al, 2011;Krause & Cantley, 2005;Pleniceanu et al, 2010). The ability of hNPC within their capacity to carry out developmental functions and unequivocally generating human renal structures encouraged us to investigate the possibility of injecting hNPCs to restore function in models of kidney failure Pleniceanu et al, 2010). Importantly, we could show beneficial effects of hNPC on improved renal function in a mouse model of chronic progressive renal injury that mimics more closely CKD in humans. Since hNPC long-term engrafted in the 5/6 Nx chronic injury model to produce kidney epithelium (even at 3 months post-hNPC injection) demonstrating regenerative potential in the remnant kidney, we hypothesize that such increases from baseline CrCl adhere at least in part to epithelial lineage differentiation of hNPCs and functional restoration. Nevertheless, tissue stem/progenitors have potential to decrease inflammation and oxidative stress and to improve angiogenesis processes altered in CKD to create a proper renal microenvironment (Yasuda et al, 2010). With the level of integration observed delineation of the molecular mediators and functional effectors of hNPC is required to assess the relative contribution of differentiation-dependent and -independent mechanisms in CKD. Altogether, while our data suggest a reparative potency for hNPC and provides cautions optimism for proceeding with allogeneic renal stem/progenitor cell therapy (ideal for clinical scenario in which CKD patients are already subscribed with an immunosuppressive drug), the absence of a significant improvement in changed CrCl following the first and second injections of hNPC and the improvement observed at 10 weeks (after three injections) suggest that in the chronic scenario sustained improvement in CrCl may require repeated injections leading to cumulative effects of hNPCs rather than single administration. These results imply the anticipated use of large cell numbers and highlight the importance in developing tools for more robust clonogenic self-renewal of hNPC and ex vivo expansion. In this regard, BMP7 showed a positive effect on hNPC growth in accordance with data in mice (Blank et al, 2009;Dudley et al, 1999). Nevertheless, deciphering additional factors to further maintain committed nephron stem/progenitors (within the NCAM1 þ fraction) is warranted. Finally, as congenital human kidney malformations are fundamentally disorders of the renal progenitor pool, cultured hNPCs with their ability to engraft and regenerate in hosts may provide new opportunities for functional analysis and disease modelling of novel inherited mutations leading to abnormal nephrogenesis and human renal developmental malformations (Vivante et al, 2013).

Research Article
www.embomolmed.org Renal developmental progenitors

Ethics statement
This study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Institutional Review Boards of Sheba Medical Center, Wolfson Hospital and Asaf Harofeh Medical Center. All pregnant women involved in the study provided written informed consent for the collection of samples from their aborted foetuses and subsequent analysis.

Immunoflorescence (IF) staining of mouse embryonic kidney organ cultures
Kidneys from E11.5 CD1 embryos were dissected and cultured on transwell membrane plates (Corning) for 4 days in Eagle's MEM (Sigma) with 10% FCS and pen/strep. Cultured kidneys were fixed in 4% PFA and antibody stained. Primary antibodies for Six2 (LifeSpan Biosciences), Cdh1 (BD transduction Laboratories) and Ncam1 (Sigma) and secondary antibodies used are summarized in Supporting Information Table S5. Images were obtained using Nikon A1R confocal microscope with and processed in ImageJ/Fiji software.

IF staining of cells
Cells were fixed with 2% PFA with 3% sucrose in PBS (for SIX2 primary antibody) or with 4% PFA (for vimentin and E-cadherin) for 10 min, and washed with PBS. Cells were blocked with 5% human serum, 5% donkey serum and 1% BSA in PBS-Tween (0.05%) followed by incubation a primary antibodies for SIX2 (Novus), vimentin (Abcam) or E-cadherin (Cell Signaling) (see Supporting Information Table S5) for 1 h in room temperature. Cells were washed and then incubated with a secondary antibody for 1 h in room temperature. Mounting containing DAPI (Dapi Fluoromount-G; SouthernBiotech, 0100-20) was applied. Images were obtained by Olympus BX51 fluorescence microscope using Olympus DP72 camera and cellSens standard software.
For comparative analysis between the fluorescence signal of NCAM1 sorted cells, images were acquired using identical settings (exposure and gain setting) using Zeiss AxioVision Software Package and analysed using ImageJ software. Signal quantification was calculated for each nucleus by determining the nuclei area and quantization of the signal in that area. The corrected total cell fluorescence (CTCF) was calculated as: integrated density À (area of selected cell Â mean fluorescence of background reading).

Flow cytometry (FACS)
1 Â 10 5 Cells were suspended in FACS buffer (0.5% BSA, 2 mM EDTA in 1Â PBS) and blocked with FcR blocking reagent (MiltenyiBiotec) and human serum (1:1). The cells were then incubated with a primary antibody or an isotype control (summarized in Supporting Information Table S3). Cells were incubated with a secondary antibody if needed (summarized in Supporting Information Table S4). Cell viability was tested using 7AAD viability staining solution (eBioscience). Cell labelling was detected using FACSCalibur (BD Pharmingen). FACS results were analysed using FlowJo analysis software.

Magnetic cell sorting
Cultured hFK cells were collected, transferred through a 30 mm preseparation filter (Miltenyi Biotec GmbH) and then washed and resuspended in pH 7.2 FACS buffer. Cells were magnetically labelled with CD56 (NCAM1) magneticbeads (Miltenyi Biotec GmbH) according to the manufacturer's instructions. Positive labelled cells were enriched on LS columns (Miltenyi Biotec GmbH) according to the manufacturer's instructions. Cell enrichment was validated using FACS.

Comparative real-time RT-PCR analysis of gene expression
Total RNA was isolated using RNeasy micro or mini kits (Qiagen GmbH) according to the manufacturer's instructions. cDNA was synthesized using the high capacity cDNA reverse transcription kit (ABI). Real-time RT-PCR (qRT-PCR) was performed using the ABI7900HT sequence detection system (Perkin-Elmer/Applied Biosystems) in the presence of TaqMan Gene Expression Master Mix or Universal PCR Master Mix (both from ABI). qRT-PCR amplification was performed using TaqMan Gene Expression Assay using StepOne plus real time RT-PCR system (ABI). hHPRT1 was used as endogenous control. The results were analysed using StepOne Software v2.1 in the DDC T (such that the expression levels of the calibrator samples ¼ 1). Statistical analysis was performed using a non-paired 2-tailed t-test. Statistical significance was considered to be p-value <0.05.

Renal differentiation of cells on chick chorio allantoic membrane (CAM)
Fertile chicken eggs were obtained from a commercial supplier and incubated at 37°C at 60-70% humidity in a forced-draft incubator. hFK cells cultured in SFM (P2) were sorted to NCAM1 þ and NCAM1 À subpopulations using magnetic beads, as described above. Cells were grafted on the CAM, as described previously Noiman et al, 2011). On Day 9 or 10 of incubation, 1 Â 10 5 or 5 Â 10 5 cells/egg suspended in 50 ml Matrigel (BD Biosciences) were pipetted into an approximately 0.8 cm plastic ring placed on the CAM (five replicates). The eggs were then incubated for an additional 5-7 days. Cell grafts were photographed after removal using a Scion Corporation colour digital camera (model CFW-1612C) attached to an Olympus SZX12 (model SZX-ILLB200). Grafts were fixed overnight in 4% buffered paraformaldehyde and embedded in paraffin. Three grafts were sampled from each treatment group for haematoxylin and eosin (H&E) staining, and one was also used for immunohistochemical (IHC) and immuofluorescence (IF) staining.

H&E staining of paraffin-embedded CAM grafts
Six micrometre sections of paraffin-embedded CAM grafts were mounted on super frost/plus glass and incubated at 60°C for 40 min. After deparaffinization, slides were incubated in Mayer's haematoxylin solution (Sigma-Aldrich) and incubated with 1% HCl in 70% ethanol for 1 min. Slides were then incubated for 10 s in eosin (Sigma-Aldrich). Images were produced using Olympus BX51TF and Scion colour and monochrome digital cameras.

IHC and IF staining of paraffin-embedded CAM grafts
Graft sections were mounted on super frost/plus glass, as described above, and deparaffinized. After 15 min of incubation in 3% H 2 O 2 in methanol, antigen retrieval (only in THG staining) was performed by boiling the slides for 15 min in a 0.01 M sodium citrate buffer (using a microwave oven). Slides were then blocked with 1% BSA in PBS for 15 min and incubated overnight at 4°C with a primary antibody (see Supporting Information Table S5). In the negative control samples, the primary antibody was substitute with non-immune serum. When IHC staining was carried out, a biotinylated second antibody was applied for 1 h, followed by incubation with horseradish peroxidase-conjugated streptavidin (HRP-SA) for 10 min. The immunoreaction was visualized by an HRP-based chromogen/substrate system, including DAB (brown) chromogen (liquid DAB substrate kit-Zymed) according to the manufacturer's instructions. The sections were then counterstained with Mayer's haematoxylin solution, dehydrated and mounted for microscopic examination. Images were obtained using a Scion Corporation grayscale digital camera (model CFW-1312M) attached to an Olympus BX51 (model BX51TF). When IF staining was performed slides were incubated for 1 h with a secondary antibody (see Supporting Information Table S5) at room temperature and counterstained with 0.1 mg/ml Hoechst in PBS for 5 min. Slides were then coverslipped in an anti-fade medium [90% glycerol (Sigma-Aldrich) with 10% PBS and 1% nPropyl gallate (Sigma-Aldrich)]. Images were obtained using an Olympus AX70 motorized microscope, and spectral unmixing using a multispectral imaging system (NuanceFX camera and software) was applied, as described previously  due to the strong autofluorescence present in both the grafted tissue and the hFK sections.
The paper explained PROBLEM: The kidney is an essential organ in mammals, which removes waste products and blood and regulates intravascular volume, electrolyte and acid/base homeostasis. Kidney disease is increasing at high rates. At present, dialysis and transplantation remain the only treatment options. Nevertheless, the shortage of available organs for transplantation continues to severely limit the latter option. Thus, there exists a tremendous clinical need to generate/construct/repair nephrons, the functional filtration unit of the kidney, and improve renal function. Regenerative medicine relies on cell transplantation, materials science, and biomedical engineering to develop biologic substitutes that can restore and maintain normal function of damaged or lost tissues and organs. We and others have been investigating the possibility of injecting functional cells to induce renal regeneration in models of kidney failure. The first challenge is to identify a stem cell capable of generation of renal components. Neither haematopoietic stem cells (HSCs) nor mesenchymal stem cells (MSCs), the most accessible human stem cells, can be used to derive true renal progenitors. Given this lack of an easy source for renal stem cells, we looked for an alternative source.

RESULTS:
Formation of kidney tissue during embryonic development requires the generation of kidney precursor cells and their subsequent differentiation into functional nephron units.
Therefore, a source for renal progenitor cells is the developing kidney. We therefore went on to identify stem/progenitor cells from within this tissue. Using defined serum free culture conditions that were adopted from studies on epithelial stem cells and specifically stimulate growth of the renal epithelial lineage and immunosorting according to NCAM1, we succeeded for the first time in prospectively isolating mitotically active human epithelial nephron progenitors (hNPCs). We show that hNPCs have primitive characteristics expressing distinct nephron progenitor markers and are highly clonogenic. Moreover, when grafted in aggregates onto the chorioallantoic membrane (CAM) of the chick embryo these cells proved robustly to generate renal structures. Collectively, foetal NCAM þ cells exhibited broad nephron epithelial differentiation (proximal, loop of Henle and distal tubules (data not shown), while in contrast grafting of control MSCs leads to generation of a disorganized cell mass. Finally we show the beneficial effects of hNPCs in a mouse model of chronic renal injury that mimics a common clinical condition in humans harboring chronic kidney disease (CKD). IMPACT: The identification of hNPCs represents advancement in stem cell science and renal medicine and paves the way to developing novel renal regenerative therapies in humans.

5/6-Nephrectomy model of chronic renal disease in NOD-SCID mice
Mice at 9-12 weeks of age were subjected to two-step 5/6 nephrectomy under halothane anaesthesia (Kasiske et al, 1988;Kennedy et al, 2003). This model was used to evaluate the therapeutic properties of hNPCs. One week after the second session, 5 Â 10 5 cells were injected directly into the parenchyma of the remnant kidney in three separate injections at intervals of 3 weeks between injections. Two weeks after each cell injection, 24 h urine samples were collected using metabolic cages and blood samples were collected from the retro-orbital sinus. On the 10th week mice were fed by high protein food followed by a fourth blood and urine sample collection. Fourteen weeks after the second Nx session, the mice were sacrificed and the kidneys were removed for IHC analysis. CrCl in ml/min was calculated using the following formula: CrCl ¼ U Cr Â 24h volume P Cr Â 24 Â 60 min IHC and IF staining of paraffin-embedded human and mouse kidney tissues Sections were pre-treated using OmniPrep solution (Zytomed Systems) in 95°C for 1 h according to manufacturer's protocol. Blocking was performed using Cas-Block solution (Invitrogen) for 20 min followed by 1 h of incubation with a primary antibodies (Cytokeratin, HLA, EMA, EnPEP or NCAM1; see Supporting Information Table S5). Samples were incubated in secondary antibody (see Supporting Information Table S5) for 30 min in room temperature and detected using DAB kit (Cell Marque) or Red Substrate Kit (Vector), both according to manufacturers' protocols. Haematoxylin was used for counterstaining. For IF staining, sections were blocked with 5% human serum, 5% donkey serum and 1% BSA in PBS-Tween (0.05%) for 1 h followed by incubation with the primary antibody for HLA (Supporting Information  Table S5) and then washed and incubated with secondary antibody (Supporting Information