RESUMO
Parkinson's disease is a degenerative brain disorder characterized by dopamine neuronal degeneration and dopamine transporter loss. In this study, we generated an induced pluripotent stem cell (iPSC) line, KNIHi001-A, from the peripheral blood mononuclear cells (PBMCs) of a 76-year-old man with Parkinson's disease. The non-integrating Sendai virus was used to reprogram iPSCs. iPSCs exhibit pluripotent markers, a normal karyotype, viral clearance, and the ability to differentiate into the three germ layers.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Masculino , Humanos , Idoso , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/metabolismo , Leucócitos Mononucleares/metabolismo , Camadas Germinativas/metabolismo , Vírus Sendai/genética , Reprogramação Celular , Diferenciação Celular/fisiologiaRESUMO
OBJECTIVES: The derivation of neural crest stem cells (NCSCs) from human pluripotent stem cells (hPSCs) has been commonly induced by WNT activation in combination with dual-SMAD inhibition. In this study, by fine-tuning BMP signalling in the conventional dual-SMAD inhibition, we sought to generate large numbers of NCSCs without WNT activation. MATERIALS AND METHODS: In the absence of WNT activation, we modulated the level of BMP signalling in the dual-SMAD inhibition system to identify conditions that efficiently drove the differentiation of hPSCs into NCSCs. We isolated two NCSC populations separately and characterized them in terms of global gene expression profiles and differentiation ability. RESULTS: Our modified dual-SMAD inhibition containing a lower dose of BMP inhibitor than that of the conventional dual-SMAD inhibition drove hPSCs into mainly NCSCs, which consisted of HNK+ p75high and HNK+ p75low cell populations. We showed that the p75high population formed spherical cell clumps, while the p75low cell population generated a 2D monolayer. We detected substantial differences in gene expression profiles between the two cell groups and showed that both p75high and p75low cells differentiated into mesenchymal stem cells (MSCs), while only p75high cells had the ability to become peripheral neurons. CONCLUSIONS: This study will provide a framework for the generation and isolation of NCSC populations for effective cell therapy for peripheral neuropathies and MSC-based cell therapy.
Assuntos
Diferenciação Celular/fisiologia , Crista Neural/citologia , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Humanos , Células-Tronco Neurais/citologia , Doenças do Sistema Nervoso Periférico/patologia , Transdução de Sinais/fisiologiaRESUMO
Successful cell therapy for Parkinson's disease (PD) requires large numbers of homogeneous ventral mesencephalic dopaminergic (vmDA) precursors. Enrichment of vmDA precursors via cell sorting is required to ensure high safety and efficacy of the cell therapy. Here, using LMX1A-eGFP knock-in reporter human embryonic stem cells, we discovered a novel surface antigen, trophoblast glycoprotein (TPBG), which was preferentially expressed in vmDA precursors. TPBG-targeted cell sorting enriched FOXA2+LMX1A+ vmDA precursors and helped attain efficient behavioral recovery of rodent PD models with increased numbers of TH+, NURR1+, and PITX3+ vmDA neurons in the grafts. Additionally, fewer proliferating cells were detected in TPBG+ cell-derived grafts than in TPBG- cell-derived grafts. Our approach is an efficient way to obtain enriched bona fide vmDA precursors, which could open a new avenue for effective PD treatment.
RESUMO
Human embryonic stem cells (hESCs) hold promise in regenerative medicine but allogeneic immune rejections caused by highly polymorphic human leukocyte antigens (HLAs) remain a barrier to their clinical applications. Here, we used a CRISPR/Cas9-mediated HLA-editing strategy to generate a variety of HLA homozygous-like hESC lines from pre-established hESC lines. We edited four pre-established HLA-heterozygous hESC lines and created a mini library of 14 HLA-edited hESC lines in which single HLA-A and HLA-B alleles and both HLA-DR alleles are disrupted. The HLA-edited hESC derivatives elicited both low T cell- and low NK cell-mediated immune responses. Our library would cover about 40% of the Asian-Pacific population. We estimate that HLA-editing of only 19 pre-established hESC lines would give rise to 46 different hESC lines to cover 90% of the Asian-Pacific population. This study offers an opportunity to generate an off-the-shelf HLA-compatible hESC bank, available for immune-compatible cell transplantation, without embryo destruction. Graphical Abstract.