A widely adaptable approach to generate integration-free iPSCs from non-invasively acquired human somatic cells.
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ABSTRACT:
SUBMITTER: Ding Z
PROVIDER: S-EPMC4417681 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:PurposeKlinefelter syndrome (KS) (47, XXY) is the most common sex chromosome abnormality in humans. KS is characterized by gynecomastia, tall stature, small testes, low testosterone levels, learning disabilities, and behavioral problems. KS is also associated with infertility due to non-obstructive azoospermia (NOA). The mechanism underlying NOA is still poorly understood, and although there is no current treatment, the use of microdissection testicular sperm extraction (micro-TESE) followed by in vitro fertilization can result in successful conception. The generation of induced pluripotent stem (iPS) cells derived from KS patients may be useful for studying the disease mechanism and identifying novel therapies.MethodsCells from a KS patient were transduced with Sendai viral vectors encoding four transcription factors, OCT4, SOX2, KLF4, and C-MYC, and the transduced cells were analyzed for in vitro and in vivo pluripotency.ResultsKS patient-derived iPS cells were successfully generated and shown to produce teratomas in the testes of SCID mice. In vitro differentiation of the iPS cells into cardiomyocyte-like cells was confirmed by the presence of clusters of beating cells.ConclusionsKS patient-derived iPS cells that could differentiate into cardiomyocyte-like cells were established.
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Project description:As a potentially unlimited autologous cell source, patient induced pluripotent stem cells (iPSCs) provide great capability for tissue regeneration, particularly in spinal cord injury (SCI). However, despite significant progress made in translation of iPSC-derived neural progenitor cells (NPCs) to clinical settings, a few hurdles remain. Among them, non-invasive approach to obtain source cells in a timely manner, safer integration-free delivery of reprogramming factors, and purification of NPCs before transplantation are top priorities to overcome. In this study, we developed a safe and cost-effective pipeline to generate clinically relevant NPCs. We first isolated cells from patients' urine and reprogrammed them into iPSCs by non-integrating Sendai viral vectors, and carried out experiments on neural differentiation. NPCs were purified by A2B5, an antibody specifically recognizing a glycoganglioside on the cell surface of neural lineage cells, via fluorescence activated cell sorting. Upon further in vitro induction, NPCs were able to give rise to neurons, oligodendrocytes and astrocytes. To test the functionality of the A2B5+ NPCs, we grafted them into the contused mouse thoracic spinal cord. Eight weeks after transplantation, the grafted cells survived, integrated into the injured spinal cord, and differentiated into neurons and glia. Our specific focus on cell source, reprogramming, differentiation and purification method purposely addresses timing and safety issues of transplantation to SCI models. It is our belief that this work takes one step closer on using human iPSC derivatives to SCI clinical settings.
| S-EPMC5629634 | biostudies-literature
Project description:Mesenchymal stem cells (MSCs) are considered a potential autologous therapy for tissue engineering. The available procedures for MSC retrieval from patients are invasive, and their limited in vitro proliferation restricts their use in the treatment of damaged tissues. Therefore, it is important to establish an alternative and safe source of MSCs. The objective of this study was to demonstrate induced pluripotent stem cell (iPSC) generation from a combination of an accessible source tissue and an integration-free method; we also attempted the differentiation of iPSCs into MSC-like cells (MSLCs) for future autologous tissue engineering. iPSCs were derived from human gingival tissues, which are easily accessible in the field of dentistry, via the use of non-integrating episomal plasmids. Established iPSCs expressed embryonic stem (ES) cell-specific markers, as assessed by gene analysis and immunocytochemistry. Embryoid bodies and teratoma formation were formed from iPSCs, showing their capacity to differentiate into three germ layers. Furthermore, we were successful in differentiating iPSCs into MSLCs. They tested positively for their capacity of trilineage differentiation. Our results demonstrate that human gingival integration-free iPSCs, readily accessible stem cells generated using episomal plasmid vectors, are a promising source of MSLCs, which can be used in tissue regeneration.
| S-EPMC4490513 | biostudies-literature
Project description:Fanconi anaemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow (BM) failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration free-induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA-deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA patient BM cells.
| S-EPMC4291073 | biostudies-literature
Project description:Induced pluripotent stem cells (iPSCs) are capable of providing an unlimited source of cells from all three germ layers and germ cells. The derivation and usage of iPSCs from various animal models may facilitate stem cell-based therapy, gene-modified animal production, and evolutionary studies assessing interspecies differences. However, there is a lack of species-wide methods for deriving iPSCs, in particular by means of non-viral and non-transgene-integrating (NTI) approaches. Here, we demonstrate the iPSC derivation from somatic fibroblasts of multiple mammalian species from three different taxonomic orders, including the common marmoset (Callithrix jacchus) in Primates, the dog (Canis lupus familiaris) in Carnivora, and the pig (Sus scrofa) in Cetartiodactyla, by combinatorial usage of chemical compounds and NTI episomal vectors. Interestingly, the fibroblasts temporarily acquired a neural stem cell-like state during the reprogramming. Collectively, our method, robustly applicable to various species, holds a great potential for facilitating stem cell-based research using various animals in Mammalia.
| S-EPMC8072067 | biostudies-literature
Project description:To elucidate the transcriptional and epigenetic alterations underlying the neurogenic defects of FA-NSCs, we conducted gene expression microarray analysis and global DNA methylation profiling. The gene expression pattern of gene-corrected NSCs (C-FA-NSCs) resembled that of control-NSCs but clustered distantly from FA-NSCs (Fig. 6F and Table S1). Hierarchical clustering based on DNA methylation levels in the promoter region (+/-1.5kb from TSS) of genes whose expression levels were rescued in C-FA-NSCs, placed C-FA-NSCs closer to control-NSCs and away from FA-NSCs (Fig. 6G), although this pattern was not seen at the whole genome level (Fig. S4C). This suggests that FANCA gene correction leads to specific methylation changes in a subset of promoters. Examination of the methylomes of NSCs derived from Fanconi Anemia iPSCs before and after gene correction by targeted bisulfite sequencing with padlock probes
2014-05-15 | E-GEOD-57685 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2014-05-21 | E-GEOD-57828 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below.
2014-05-21 | GSE57828 | GEO
Project description:To elucidate the transcriptional and epigenetic alterations underlying the neurogenic defects of FA-NSCs, we conducted gene expression microarray analysis and global DNA methylation profiling. The gene expression pattern of gene-corrected NSCs (C-FA-NSCs) resembled that of control-NSCs but clustered distantly from FA-NSCs (Fig. 6F and Table S1). Hierarchical clustering based on DNA methylation levels in the promoter region (+/-1.5kb from TSS) of genes whose expression levels were rescued in C-FA-NSCs, placed C-FA-NSCs closer to control-NSCs and away from FA-NSCs (Fig. 6G), although this pattern was not seen at the whole genome level (Fig. S4C). This suggests that FANCA gene correction leads to specific methylation changes in a subset of promoters.
2014-05-15 | GSE57685 | GEO
Project description:Induced pluripotent stem cells (iPSCs) give rise to neural stem/progenitor cells, serving as a good source for neural regeneration. Here, we established transgene-free (TF) iPSCs from dental stem cells (DSCs) and determined their capacity to differentiate into functional neurons in vitro. Generated TF iPSCs from stem cells of apical papilla and dental pulp stem cells underwent two methods-embryoid body-mediated and direct induction, to guide TF-DSC iPSCs along with H9 or H9 Syn-GFP (human embryonic stem cells) into functional neurons in vitro. Using the embryoid body-mediated method, early stage neural markers PAX6, SOX1, and nestin were detected by immunocytofluorescence or reverse transcription-real time polymerase chain reaction (RT-qPCR). At late stage of neural induction measured at Weeks 7 and 9, the expression levels of neuron-specific markers Nav1.6, Kv1.4, Kv4.2, synapsin, SNAP25, PSD95, GAD67, GAP43, and NSE varied between stem cells of apical papilla iPSCs and H9. For direct induction method, iPSCs were directly induced into neural stem/progenitor cells and guided to become neuron-like cells. The direct method, while simpler, showed cell detachment and death during the differentiation process. At early stage, PAX6, SOX1 and nestin were detected. At late stage of differentiation, all five genes tested, nestin, ?III-tubulin, neurofilament medium chain, GFAP, and Nav, were positive in many cells in cultures. Both differentiation methods led to neuron-like cells in cultures exhibiting sodium and potassium currents, action potential, or spontaneous excitatory postsynaptic potential. Thus, TF-DSC iPSCs are capable of undergoing guided neurogenic differentiation into functional neurons in vitro, thereby may serve as a cell source for neural regeneration.
| S-EPMC6482049 | biostudies-literature