RESUMO
Human pluripotent stem cells (hPSCs) provide a source for the generation of defined kidney cells and renal organoids applicable in regenerative medicine, disease modeling, and drug screening. These applications require the provision of hPSC-derived renal cells by reproducible, scalable, and efficient methods. We established a chemically defined protocol by application of Activin A, BMP4, and Retinoic acid followed by GDNF, which steered hPSCs to the renal lineage and resulted in populations of SIX2+/CITED1+ metanephric mesenchyme- (MM) and of HOXB7+/GRHL2+ ureteric bud (UB)-like cells already by 6 days. Transcriptome analysis corroborated that the PSC-derived cell types at day 8 resemble their renal vesicle and ureteric epithelial counterpart in vivo, forming tubular and glomerular renal cells 6 days later. We demonstrate that starting from hPSCs, our in vitro protocol generates a pool of nephrogenic progenitors at the renal vesicle stage, which can be further directed into specialized nephronal cell types including mesangial-, proximal tubular-, distal tubular, collecting duct epithelial cells, and podocyte precursors after 14 days. This simple and rapid method to produce renal cells from a common precursor pool in 2D culture provides the basis for scaled-up production of tailored renal cell types, which are applicable for drug testing or cell-based regenerative therapies.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Néfrons/citologia , Células-Tronco Pluripotentes/citologia , Ativinas/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Humanos , Néfrons/efeitos dos fármacos , Néfrons/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Transcriptoma/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
Individuals with transient reception potential cation channel 6 (TRPC6) mutation have variable phenotypes, ranging from healthy carriers to focal segmental glomerulosclerosis (FSGS). Human induced pluripotent stem cell (hiPSC) line was generated from the urinary cells of a patient with FSGS with a mutant variant of TRPC6. The cells were reprogrammed with Yamanaka factors (OCT3, SOX2, LIN28, L-MYC, and KLF4) using a commercially available Epi5 Reprogramming Kit. The pluripotency of the hiPSC line was confirmed by the expression of common stem cell markers and by their ability to generate all germ layers in vitro. The line is available and registered in the human pluripotent stem cell registry as BCRTi006-A. The generated line represents a valuable tool for disease modeling and drug development for FSGS.
Assuntos
Glomerulosclerose Segmentar e Focal , Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Glomerulosclerose Segmentar e Focal/genética , Canal de Cátion TRPC6/genética , Canal de Cátion TRPC6/metabolismo , Mutação , Diferenciação Celular , Reprogramação CelularRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder of adults, characterized by uncontrolled cysts formation that causes a gradual impairment of kidney function. We generated a human induced pluripotent stem cell (hiPSC) line from the urinary cells of a patient diagnosed with ADPKD using a non-integrating Epi5™ Episomal iPSC reprogramming strategy. Characterization of the cell line was performed regarding their undifferentiated status, differentiation potential, and quality control for karyotypic integrity, identity, and clearance of reprogramming vectors. The newly derived hiPSC line, namely BCRTi007-A, can be used in vitro for disease modeling of ADPKD as well as testing for novel therapeutic approaches.
Assuntos
Células-Tronco Pluripotentes Induzidas , Rim Policístico Autossômico Dominante , Adulto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Mutação , Diferenciação Celular , Linhagem CelularRESUMO
The success of human induced pluripotent stem cell (hiPSC)-based therapy critically depends on understanding and controlling the immunological effects of the hiPSC-derived transplant. While hiPSC-derived cells used for cell therapy are often immature with post-grafting maturation, immunological properties may change, with adverse effects on graft tolerance and control. In the present study, the allogeneic and autologous cellular immunity of hiPSC-derived progenitor and terminally differentiated cells were investigated in vitro. In contrast to allogeneic primary cells, hiPSC-derived early renal progenitors and mature renal epithelial cells are both tolerated not only by autologous but also by allogeneic T cells. These immune-privileged properties result from active immunomodulation and low immune visibility, which decrease during the process of cell maturation. However, autologous and allogeneic natural killer (NK) cell responses are not suppressed by hiPSC-derived renal cells and effectively change NK cell activation status. These findings clearly show a dynamic stage-specific dependency of autologous and allogeneic T and NK cell responses, with consequences for effective cell therapies. The study suggests that hiPSC-derived early progenitors may provide advantageous immune-suppressive properties when applied in cell therapy. The data furthermore indicate a need to suppress NK cell activation in allogeneic as well as autologous settings.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Células Matadoras Naturais , Ativação LinfocitáriaRESUMO
OBJECTIVE: To provide a standardized protocol for large-scale production of proximal tubular epithelial cells (PTEC) generated from human pluripotent stem cells (hPSC). METHODS: The hPSC were expanded and differentiated into PTEC on matrix-coated alginate beads in an automated levitating fluidic platform bioLevitator. Differentiation efficacy was evaluated by immunofluorescence staining and flow cytometry, ultrastructure visualized by electron microscopy. Active reabsorption by PTEC was investigated by glucose, albumin, organic anions and cations uptake assays. Finally, the response to cisplatin-treatment was assessed to check the potential use of PTEC to model drug-induced nephrotoxicity. RESULTS: hPSC expansion and PTEC differentiation could be performed directly on matrix-coated alginate beads in suspension bioreactors. Renal precursors arose 4 days post hPSC differentiation and PTEC after 8 days with 80% efficiency, with a 10-fold expansion from hPSC in 24 days. PTEC on beads, exhibited microvilli and clear apico-basal localization of markers. Functionality of PTECs was confirmed by uptake of glucose, albumin, organic anions and cations and expression of KIM-1 after Cisplatin treatment. CONCLUSION: We demonstrate the efficient expansion of hPSC, controlled differentiation to renal progenitors and further specification to polarized tubular epithelial cells. This is the first report employing biolevitation and matrix-coated beads in a completely defined medium for the scalable and potentially automatable production of functional human PTEC.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Meios de Cultura , Células Epiteliais/metabolismo , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Glucose/metabolismo , Humanos , Túbulos Renais Proximais/citologiaRESUMO
Native extracellular matrix (ECM) provides scaffolds for tissue engineering with natural architecture and biochemical composition. Maintaining the native ECM in decellularized tissues provides cues for cells, which promote their tissue specific arrangement and function. Several approaches have been used to decellularize ECM from the kidney in order to reestablish renal tissue but their comparability is hampered because methods for decellularization and assessment of ECM vary widely. Therefore, we applied a standardized immersion protocol to decellularize porcine kidney tissue with three detergents Triton X-100, SDS and sodium deoxycholate (SDC) at variable temperatures. For comparative analysis decellularization efficacies, structural preservation, composition and cell attachment and viability were analyzed. Structural ECM-conservation is strongly dependent on decellularization temperature, while preservation of glycosaminoglycans (GAG), collagens and cytokines was affected by the detergents used. GAG and collagens were best maintained by 1% SDS at 4 °C, whereas cytokines were best maintained in 1% SDC at 4 °C. Viability and attachment of human induced pluripotent stem cell derived renal precursor cells were best in SDC-ECM and thus not associated with the degree of GAG and collagen maintenance but the cytokine preservation. Based on structural and functional characteristics, we developed a scoring system that allows intra- and inter-study comparison of decellularization strategies. Application of the scoring system to our experimental data showed that decellularization with 1% SDS at 4 °C provided the highest structural and composition scores, while 1% SDC at 4 °C had lower structural and composition but a significantly better cell performance score. Inclusion of multiple published studies in the scoring matrix for comparison identified the highest structural and composition scores when decellularization was performed with SDS at low concentration, for a short period of time and at low temperature. Furthermore, the scoring system indicated that cell attachment and viability cannot be concluded from any other parameter and should therefore always be included in evaluation of decellularization strategies.
Assuntos
Colágeno/metabolismo , Células Epiteliais/metabolismo , Matriz Extracelular/química , Glicosaminoglicanos/química , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Rim/fisiologia , Octoxinol/química , Engenharia Tecidual/métodos , Animais , Colágeno/química , Detergentes , Células Epiteliais/química , Células Epiteliais/citologia , Glicosaminoglicanos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/química , Rim/química , SuínosRESUMO
We have generated a human induced pluripotent stem cell (iPSC) line derived from urinary cells of a 28year old healthy female donor. The cells were reprogrammed using a non-integrating viral vector and have shown full differentiation potential. Together with the iPSC line, the donor provided blood cells for the study of immunological effects of the iPSC line and its derivatives in autologous and allogeneic settings. The line is available and registered in the human pluripotent stem cell registry as BCRTi005-A.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Vírus Sendai , Transdução Genética , Urina/citologia , Linhagem Celular , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
Fibrodysplasia ossificans progressiva (FOP) is an extremely rare, autosomal dominant transmitted genetic disease. Patients experience progressive bone formation replacing tendons, ligaments, muscle and soft tissue. Cause of FOP are gain-of-function mutations in the Bone Morphogenetic Protein (BMP) receptor Activin A receptor type 1 (ACVR1) (Kaplan et al., 2008). The most common mutation is R206H, which leads to the substitution of codon 206 from arginine to histidine (Shore et al., 2006). Here, we describe the derivation and characterization of two hiPSC lines from two FOP patients, both carrying the mutation R206H. Cells were isolated from urine and reprogrammed using integration free Sendai virus vectors under defined conditions.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miosite Ossificante/patologia , Miosite Ossificante/urina , Sequência de Bases , Biomarcadores/metabolismo , Diferenciação Celular , Forma Celular , Células Cultivadas , Impressões Digitais de DNA , Humanos , Cariotipagem , Reprodutibilidade dos Testes , Vírus Sendai/fisiologia , Análise de Sequência de DNARESUMO
The Publisher regrets that this article is an accidental duplication of an article that has already been published in Stem Cell Res., 16 (2016) 133136, http://dx.doi.org/10.1016/j.scr.2015.12.021. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
RESUMO
We have generated a human induced pluripotent stem cell (iPSC) line derived from urinary cells of a 30 year old healthy female donor. The cells were reprogrammed using a non-integrating viral vector and have shown full differentiation potential. Together with the iPSC-line, the donor provided blood cells for the study of immunological effects of the iPSC line and its derivatives in autologous and allogeneic settings. The line is available and registered in the human pluripotent stem cell registry as BCRTi004-A.