scholarly journals A Comprehensive Molecular Portrait of Human Urine-derived Renal Progenitor Cells

2019 ◽  
Author(s):  
Md Shaifur Rahman ◽  
Wasco Wruck ◽  
Lucas-Sebastian Spitzhorn ◽  
Martina Bohndorf ◽  
Soraia Martins ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Human Urine ◽  
Interaction Network ◽  
Self Renewal ◽  
Non Invasive ◽  
Renal Progenitor Cells ◽  
Associated Proteins ◽  
Renal Progenitor

AbstractBackgroundHuman urine is now recognised as a non-invasive source of stem cells with regeneration potential. These cells are mesenchymal stem cells but their detailed molecular and cellular identities are poorly defined. Furthermore, unlike the mouse, the gene regulatory network driving self-renewal and differentiation into functional renal cells in vitro remain unresolved.MethodsWe isolated urine stem cells from 10 individuals from both genders and distinct ages, characterized them as renal progenitor cells and explored the gene regulatory network sustaining self-renewal.ResultsThese cells express pluripotency-associated proteins-TRA-1-60, TRA-1-81, SSEA4, C-KIT and CD133. Expression of pluripotency-associated proteins enabled rapid reprogramming into iPSCs using episomal-based plasmids without pathway perturbations. Transcriptome analysis revealed expression of a plethora of nephrogenesis-related genes such as SIX2, OSR1, CITED1, NPHS2, NPHS1, PAX2, SALL1, AQP2, EYA1, SLC12A1 and UMOD. As expected, the cells transport Albumin by endocytosis. Based on this, we refer to these cells as urine derived renal progenitor cells-UdRPCs. Associated GO-term analysis of UdRPCs and UdRPC-iPSCs underlined their renal identity and functionality. Upon differentiation by WNT activation using the GSK3β-inhibitor (CHIR99021), transcriptome and KEGG pathway analysis revealed upregulation of WNT-associated genes-AXIN2, JUN and NKD1. Protein interaction network identified JUN- a downstream target of the WNT pathway in association with STAT3, ATF2 and MAPK1 as a putative regulator of self-renewal and differentiation in UdRPCs. Furthermore, like pluripotent stem cells, self-renewal is maintained by FGF2-driven TGFβ-SMAD2/3 pathway.ConclusionThis in vitro model and the data presented should lay the foundation for studying nephrogenesis in man.Significance StatementHuman urine is a non-invasive source of stem cells with regeneration potential. Here, we investigated the cellular and molecular identities, and the gene regulation driving self-renewal and differentiation of these cells in vitro. These cells express pluripotency-associated markers enabling easy reprogramming. Based on the expression of renal associated genes, proteins and functionality, we refer to these cells as urine derived renal progenitor cells-UdRPCs. CHIR99021-induced differentiation of UdRPCs activated WNT-related genes-AXIN2, JUN and NKD1. Protein interaction network identified JUN as a putative regulator of differentiation whereas self-renewal is maintained by FGF2-driven TGFβ-SMAD2/3. Our data will enhance understanding of the molecular identities of UdRPCs, and enable the generation of renal disease models in vitro and eventually kidney-associated regenerative therapies.

scholarly journals Renal Progenitor Cells Have Higher Genetic Stability and Lower Oxidative Stress than Mesenchymal Stem Cells during In Vitro Expansion

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Elís Rosélia Dutra de Freitas Siqueira Silva ◽  
Napoleão Martins Argôlo Neto ◽  
Dayseanny de Oliveira Bezerra ◽  
Sandra Maria Mendes de Moura Dantas ◽  
Lucilene dos Santos Silva ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Dna Damage ◽  
Mesenchymal Stem Cells ◽  
Progenitor Cells ◽  
Genetic Stability ◽  
Renal Progenitor Cells ◽  
In Vitro Expansion ◽  
Renal Progenitor

In vitro senescence of multipotent cells has been commonly associated with DNA damage induced by oxidative stress. These changes may vary according to the sources of production and the studied lineages, which raises questions about the effect of growing time on genetic stability. This study is aimed at evaluating the evolution of genetic stability, viability, and oxidative stress of bone marrow mesenchymal stem cells (MSCBMsu) and renal progenitor cells of the renal cortex (RPCsu) of swine (Sus scrofa domesticus) in culture passages. P2, P5, and P9 were used for MSCBMsu and P1, P2, and P3 for RPCsu obtained by thawing. The experimental groups were submitted to MTT, apoptosis and necrosis assays, comet test, and reactive substance measurements of thiobarbituric acid (TBARS), nitrite, reduced glutathione (GSH), and catalase. The MTT test curve showed a mean viability of 1.14±0.62 and 1.12±0.54, respectively, for MSCBMsu and RPCsu. The percentages of MSCBMsu and RPCsu were presented, respectively, for apoptosis, an irregular and descending behavior, and necrosis, ascending and irregular. The DNA damage index showed higher intensity among the MSCBMsu in the P5 and P9 passages (p<0.05). In the TBARS evaluation, there was variation among the lines of RPCsu and MSCBMsu, presenting the last most significant variations (p<0.05). In the nitrite values, we identified only among the lines, in the passages P1 and P2, with the highest averages displayed by the MSCBMsu lineage (p<0.05). The measurement of antioxidant system activity showed high standards, identifying differences only for GSH values, in the RPCsu lineage, in P3 (p<0.05). This study suggests that the maintenance of cell culture in the long term induces lower regulation of oxidative stress, and RPCsu presents higher genetic stability and lower oxidative stress than MSCBMsu during in vitro expansion.

scholarly journals Sall1 balances self-renewal and differentiation of renal progenitor cells

Development ◽  
2014 ◽  
Vol 141 (5) ◽  
pp. 1047-1058 ◽  
Author(s):  
J. M. Basta ◽  
L. Robbins ◽  
S. M. Kiefer ◽  
D. Dorsett ◽  
M. Rauchman
Keyword(s):  
Progenitor Cells ◽  
Self Renewal ◽  
Renal Progenitor Cells ◽  
Renal Progenitor

scholarly journals Role of bone marrow-derived stem cells, renal progenitor cells and stem cell factor in chronic renal allograft nephropathy

2013 ◽  
Vol 49 (3) ◽  
pp. 235-247
Author(s):  
Hayam Abdel Meguid El Aggan ◽  
Mona Abdel Kader Salem ◽  
Nahla Mohamed Gamal Farahat ◽  
Ahmad Fathy El-Koraie ◽  
Ghaly Abd Al-Rahim Mohammed Kotb
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Renal Allograft ◽  
Renal Progenitor Cells ◽  
Renal Progenitor

scholarly journals Constructing an Isogenic 3D Human Nephrogenic Progenitor Cell Model Composed of Endothelial, Mesenchymal, and SIX2-Positive Renal Progenitor Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Lisa Nguyen ◽  
Lucas-Sebastian Spitzhorn ◽  
James Adjaye
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Progenitor Cells ◽  
Pluripotent Stem Cells ◽  
Progenitor Cell ◽  
Cell Model ◽  
Renal Progenitor Cells ◽  
Renal Stem Cells ◽  
Renal Progenitor

Urine has become the source of choice for noninvasive renal epithelial cells and renal stem cells which can be used for generating induced pluripotent stem cells. The aim of this study was to generate a 3D nephrogenic progenitor cell model composed of three distinct cell types—urine-derived SIX2-positive renal progenitor cells, iPSC-derived mesenchymal stem cells, and iPSC-derived endothelial cells originating from the same individual. Characterization of the generated mesenchymal stem cells revealed plastic adherent growth and a trilineage differentiation potential to adipocytes, chondrocytes, and osteoblasts. Furthermore, these cells express the typical MSC markers CD73, CD90, and CD105. The induced endothelial cells express the endothelial cell surface marker CD31. Upon combination of urine-derived renal progenitor cells, induced mesenchymal stem cells, and induced endothelial cells at a set ratio, the cells self-condensed into three-dimensional nephrogenic progenitor cells which we refer to as 3D-NPCs. Immunofluorescence-based stainings of sectioned 3D-NPCs revealed cells expressing the renal progenitor cell markers (SIX2 and PAX8), podocyte markers (Nephrin and Podocin), the endothelial marker (CD31), and mesenchymal markers (Vimentin and PDGFR-β). These 3D-NPCs share kidney progenitor characteristics and thus the potential to differentiate into podocytes and proximal and distal tubules. As urine-derived renal progenitor cells can be easily obtained from cells shed into urine, the generation of 3D-NPCs directly from renal progenitor cells instead of pluripotent stem cells or kidney biopsies holds a great potential for the use in nephrotoxicity tests, drug screening, modelling nephrogenesis and diseases.

scholarly journals WNT/β-Catenin Signaling Is Required for Integration of CD24+Renal Progenitor Cells into Glycerol-Damaged Adult Renal Tubules

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Zhao Zhang ◽  
Diana M. Iglesias ◽  
Rachel Corsini ◽  
LeeLee Chu ◽  
Paul Goodyer
Keyword(s):  
Progenitor Cells ◽  
Kidney Injury ◽  
Perinatal Period ◽  
Renal Tubules ◽  
Adult Mice ◽  
Renal Progenitor Cells ◽  
Nephron Progenitor ◽  
Canonical Wnt ◽  
Renal Progenitor

During development, nephron progenitor cells (NPC) are induced to differentiate by WNT9b signals from the ureteric bud. Although nephrogenesis ends in the perinatal period, acute kidney injury (AKI) elicits repopulation of damaged nephrons. Interestingly, embryonic NPC infused into adult mice with AKI are incorporated into regenerating tubules. Since WNT/β-catenin signaling is crucial for primary nephrogenesis, we reasoned that it might also be needed for the endogenous repair mechanism and for integration of exogenous NPC. When we examined glycerol-induced AKI in adult mice bearing aβ-catenin/TCF reporter transgene, endogenous tubular cells reexpressed the NPC marker, CD24, and showed widespreadβ-catenin/TCF signaling. We isolated CD24+cells from E15 kidneys of mice with the canonical WNT signaling reporter. 40% of cells responded to WNT3ain vitroand when infused into glycerol-injured adult, the cells exhibitedβ-catenin/TCF reporter activity when integrated into damaged tubules. When embryonic CD24+cells were treated with aβ-catenin/TCF pathway inhibitor (IWR-1) prior to infusion into glycerol-injured mice, tubular integration of cells was sharply reduced. Thus, the endogenous canonicalβ-catenin/TCF pathway is reactivated during recovery from AKI and is required for integration of exogenous embryonic renal progenitor cells into damaged tubules. These events appear to recapitulate the WNT-dependent inductive process which drives primary nephrogenesis.

Models of Pluripotent and Somatic Stem Cells to Study Tissue-Specific Sensitivities in Fanconi Anemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 168-168 ◽  
Author(s):  
Timothy Michael Chlon ◽  
Susanne I Wells ◽  
Sonya Ruiz-Torres ◽  
Matthew Kuhar ◽  
James M Wells
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Progenitor Cells ◽  
Fanconi Anemia ◽  
Squamous Epithelium ◽  
Somatic Stem Cells ◽  
Self Renewal ◽  
Stem And Progenitor Cells ◽  
Pathway Function

Abstract The Fanconi Anemia (FA) DNA Repair pathway functions through homologous recombination for error-free repair of DNA interstrand crosslinks. Loss of function of this pathway causes a complex genetic disease that is characterized by congenital abnormalities, bone marrow failure (BMF), and extreme incidence of squamous cell carcinomas. BMF is caused by exhaustion of hematopoietic stem and progenitor cells (HSPCs) and is nearly 100% penetrant by age 40 in FA patients, indicating a profound sensitivity of HSPCs to FA pathway deficiency. In contrast, stem cells in other rapidly regenerating tissues, such as the skin and intestine, are not similarly exhausted. Interestingly, squamous epithelium is highly prone to transformation while intestinal epithelium is not. In order to explore the developmental origins of such striking tissue-specific phenotypes in FA patients, we have generated induced pluripotent stem cell (iPSC) lines conditional for FA pathway function (cFA-iPSCs) and used them to derive FA-proficient and deficient in vitro models of diverse tissues. FA patient cells are refractory to reprogramming. To circumvent this defect and prevent the selection of FA-resistant iPSC clones, fibroblasts from 2 FANCA patients were inducibly complemented with a FANCA transgene under the control of a tetracycline-inducible promoter and then were reprogrammed to iPSC. In this way, the FA pathway was functional throughout reprogramming and could then be turned on or off in established iPSC lines by the addition or withdrawal of doxycycline (DOX) to the media. Here, we describe the effect of FA pathway loss on iPSCs, and present preliminary data on iPSC-derived equivalents of three lineages: hematopoietic, squamous, and intestinal. First, functional consequences of FA pathway loss on iPSC pluripotency and self-renewal were examined. Upon withdrawal of DOX from the culture media, the complementing FA transgene was effectively silenced, resulting in loss of FA pathway function within 7 days. FA-deficient iPSCs maintained normal expression of OCT-3/4 and NANOG and formed teratomas in NSG mice, indicating that pluripotency was maintained. However, profound cell cycle arrest and apoptosis were observed under normal in vitro culture conditions within 7 days of DOX-withdrawal, and the iPSCs failed to expand by 2-3 passages. Thus, we concluded that iPSCs require an intact FA pathway for self-renewal in vitro. Mechanistic studies of FA pathway-deficient iPSCs revealed a 10-fold increase in gH2AX foci in the G2-M phase of the cell cycle. This correlated with activated DNA damage response signaling through ATR and CHK1. Inhibition of CHK1 completely restored the growth of FA-deficient iPSCs to that of their FA-proficient counterparts through a remarkable rapid bypass of the G2-M checkpoint. Unexpectedly, cells maintained in CHK1 inhibitor for over 40days accrued few karypotypic abnormalities (<5% of cells), of which most were trisomies, and only 1 cell out of 40 contained translocations. The rarity of deletions and translocations in CHK1 inhibited iPSC suggests that error-free repair still occurs by an unknown mechanism. We next differentiated the cFA-iPSCs into 3D squamous epithelium and intestine with timed-withdrawal of the FA pathway to determine the effect of FA pathway loss on tissue development and homeostasis. Squamous epithelial rafts and intestinal organoids were generated in the presence and absence of DOX using established protocols. These demonstrated that FA pathway loss does not cause gross abnormalities in epithelial tissues, in line with patient phenotypes. We will present the latest results on proliferation, survival, and differentiation of stem and progenitor cells within each tissue. These will include an assessment of epithelial hyperplasia, which has been observed previously in immortalized FA patient keratinocyte-derived organotypic squamous epithelial rafts and in the oral epithelia of FANCD2 knockout mice. Finally, we will present our latest data on the effects of FA deficiency on hematopoietic progenitor cells, which are currently being generated from the cFA-iPSCs. Collectively, these experiments will quantify cell intrinsic sensitivity of pluripotent versus somatic stem cells that reside in diverse tissue types to loss of the FA pathway. The results may inform the development of novel therapeutics to treat FA BMF without increasing disease risk in other tissues. Disclosures No relevant conflicts of interest to declare.

scholarly journals Therapeutic potential of human induced pluripotent stem cells and renal progenitor cells in experimental chronic kidney disease 

2020 ◽  
Author(s):  
Patrícia de Carvalho Ribeiro ◽  
Fernando Henrique Lojudice ◽  
Ida Maria Maximina Fernandes-Charpiot ◽  
Maria Alice Sperto Ferreira Baptista ◽  
Stanley de Almeida Araújo ◽  
...  
Keyword(s):  
Chronic Kidney Disease ◽  
Stem Cells ◽  
Kidney Disease ◽  
Cell Therapy ◽  
Induced Pluripotent Stem Cells ◽  
Progenitor Cells ◽  
Pluripotent Stem Cells ◽  
Renal Progenitor Cells ◽  
Induced Pluripotent ◽  
Renal Progenitor

Abstract BackgroundChronic Kidney Disease (CKD) is a global public health problem. Cell therapy using pluripotent stem cells represents an attractive therapeutic approach for the treatment of CKD.MethodsWe transplanted Mitomycin C (MMC)-treated human induced pluripotent stem cells (hiPSCs) and renal progenitor cells (RPCs) into a CKD rat model system. The RPCs and hiPSCs cells were characterized by immunofluorescence and qRT-PCR. Untreated 5/6 nephrectomized rats were compared to CKD animals receiving the same amount of MMC-treated hiPSCs or RPCs. Renal function, histology and immunohistochemistry were evaluated 45 days post-surgery. ResultsWe successfully generated hiPSCs from peripheral blood and differentiated them into RPCs expressing renal progenitor genes (PAX2, WT1, SIX2, and SALL1) and podocyte-related genes (SYNPO, NPHS1). RPCs also exhibited reduced OCT4 expression, confirming the loss of pluripotency. After cell transplantation into CKD rats, the body weight change was significantly increased in both hiPSC and RPC groups, in comparison with the control group. Creatinine clearance (CCr) was preserved only in the hiPSC group. Similarly, the number of macrophages in the kidneys of the hiPSC group reached a statistically significant reduction, when compared to control rats. Both treatments reduced positive staining for the marker α-smooth muscle actin. Histological features showed decreased tubulointerstitial damage (interstitial fibrosis and tubular atrophy) as well as a reduction in glomerulosclerosis in both iPSC and RPC groups.ConclusionsIn conclusion, we describe that both MMC-treated hiPSCs and RPCs exert beneficial effects in attenuating CKD progression. Both cell types were equally efficient to reduce histological damage and weight loss caused by CKD. hiPSCs seems to be more efficient than RPCs, possibly due to a paracrine effect triggered by hiPSCs. These results demonstrate that the use of MMC-treated hiPSCs and RPCs improve clinical and histological CKD parameters, avoided tumor formation, and therefore may be a promising cell therapy strategy for CKD.

Growth Factor Receptor-Bound Protein 10 (Grb10) Regulates Hematopoietic Stem Cell Renewal

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 248-248
Author(s):  
Xiao Yan ◽  
Heather A. Himburg ◽  
Phuong L. Doan ◽  
Mamle Quarmyne ◽  
Nelson J. Chao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factor ◽  
Progenitor Cells ◽  
Growth Factor Receptor ◽  
Self Renewal ◽  
Donor Cells ◽  
Growth Factor Receptor Bound

Abstract The mechanisms which regulate HSC regeneration following stress or injury remain poorly understood. Precise study of HSCs during regeneration has been impeded by the rarity of the HSC population and depletion of phenotypic HSCs early following genotoxic stresses, such as total body irradiation (TBI). We isolated bone marrow (BM) ckit+sca-1+lin- (KSL) cells, which are enriched for HSCs, from adult C57Bl6 mice before and at several time points following TBI, as a means to map the dynamic molecular response of HSC regeneration. Following 550cGy TBI, BM KSL cells were depleted by 7 days post-TBI, whereas KSL cell recovery was evident at day+14. We isolated BM KSL cells and myeloid progenitor cells (c-kit+sca-1-lin- cells) at day +14 and compared the gene expression profile of regenerating HSCs versus steady state HSCs (non-irradiated) and committed progenitor cells. We identified growth factor receptor-bound protein 10 (Grb10), a co-receptor which regulates Insulin Receptor/IGF-1 signaling, to be significantly overexpressed in regenerating BM KSL cells compared to non-irradiated KSL cells (3.3 fold, p<0.0001). Grb10 is a member of the family of imprinted genes which are predominately expressed in numerous stem cell populations, including embryonic stem cells, skin and muscle stem cells. Viral shRNA-mediated knockdown of Grb10 in BM KSL cells caused a significant decrease in KSL cells and colony forming cells (CFCs) in detected in 7 day culture (p=0.03 and p=0.002, respectively). Furthermore, mice which were competitively transplanted with Grb10-deficient KSL cells had 10-fold decreased donor, multilineage hematopoietic cell engraftment than mice transplanted with Grb10-expressing HSCs (p=0.007 for %CD45.1+ donor cells). Secondary competitive repopulation assays confirmed > 10-fold deficit in long-term repopulating capacity in Grb10 deficient KSL cells compared to Grb10 expressing KSL cells (p=0.006 for %CD45.1+ donor cells in secondary mice). In order to examine the effect of Grb10-deficiency on HSC fate and hematopoiesis in vivo, we generated maternally-derived Grb10-deficient mice. Heterozygous 8-week old Grb10m/+ (1 mutant allele, 1 wild type allele) were found to have 10-fold decreased Grb10 expression in BM lin- cells and had normal range complete blood counts. However, BM CFCs were significantly decreased in Grb10m/+ mice compared to Grb10+/+ mice (p=0.006) and competitive repopulation assays demonstrated significantly decreased donor hematopoietic cell repopulation in recipient mice transplanted with Grb10m/+ BM cells versus mice transplanted with Grb10+/+ BM cells (1/14, 7% vs. 5/14, 38% of mice with > 0.1% donor CD45.2+ cells). These results suggest that Grb10 regulates HSC self-renewal in vitro and in vivo. Mechanistically, Grb10m/+ mice displayed no alterations in the cell cycle status or frequency of apoptotic cells within BM HSCs compared to Grb10+/+ mice. However, single cytokine functional screening suggested that Grb10 regulates SCF-mediated proliferation of HSCs. Grb10m/+ BM KSL cells generated significantly less CFCs in culture in response to SCF treatment compared to Grb10+/+ KSL cells (p=0.008). Commensurate with this, SCF-mediated activation of mTOR was significantly increased in Grb10m/+ KSL cells compared to that observed in Grb10+/+ KSL cells (p=0.006). These data suggest that cytokine-mediated induction of mTOR signaling, which has been shown to deplete functional HSCs, is antagonized by Grb10, and that Grb10 is necessary to block cytokine-mediated HSC differentiation in vitro and in vivo. Grb10 represents a novel regulator of HSC fate determination and a new mechanistic target to facilitate HSC self-renewal. Studies are underway to determine whether Grb10 is also necessary for HSC regeneration after TBI. Disclosures No relevant conflicts of interest to declare.

scholarly journals The FGF, TGFβ and WNT axis Modulate Self-renewal of Human SIX2+ Urine Derived Renal Progenitor Cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Md Shaifur Rahman ◽  
Wasco Wruck ◽  
Lucas-Sebastian Spitzhorn ◽  
Lisa Nguyen ◽  
Martina Bohndorf ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Self Renewal ◽  
Renal Progenitor Cells ◽  
Renal Progenitor
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