Correction: Generation of Human Induced Pluripotent Stem (iPS) Cells in Serum- and Feeder-Free Defined Culture and TGF-β1 Regulation of Pluripotency
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PROVIDER: S-EPMC3958556 | biostudies-literature | 2014 Jan
REPOSITORIES: biostudies-literature
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Project description:Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka's factors (Oct3/4, Sox2, Klf4, and c-Myc) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo. In this study, we found that TGF-?1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-?1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-?1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.
| S-EPMC3906124 | biostudies-literature
Project description:Human stem cells are promising sources for bladder regeneration. Among several possible sources, pluripotent stem cells are the most fascinating because they can differentiate into any cell type, and proliferate limitlessly in vitro. Here, we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE), and further specified DE cells into BUCs by treating retinoic acid under a keratinocyte-specific serum free medium. hPSC-derived DE cells showed significantly expressed DE-specific genes, but did not express mesodermal or ectodermal genes. After DE cells were specified into BUCs, they notably expressed urothelium-specific genes such as UPIb, UPII, UPIIIa, P63 and CK7. Immunocytochemistry showed that BUCs expressed UPII, CK8/18 and P63 as well as tight junction molecules, E-CADHERIN and ZO-1. Additionally, hPSCs-derived BUCs exhibited low permeability in a FITC-dextran permeability assay, indicating BUCs possessed the functional units of barrier on their surfaces. However, BUCs did not express the marker genes of other endodermal lineage cells (intestine and liver) as well as mesodermal or ectodermal lineage cells. In summary, we sequentially differentiated hPSCs into DE and BUCs in a serum- and feeder-free condition. Our differentiation protocol will be useful for producing cells for bladder regeneration and studying normal and pathological development of the human bladder urothelium in vitro.
| S-EPMC4057664 | biostudies-literature
Project description:Differentiation of keratinocytes from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) has become an important tool for wound healing research and for studying skin diseases in instances where patient cells are not available. Several keratinocyte differentiation protocols using hiPSC colony fragments or embryoid bodies have been published with some requiring prolonged time for differentiation or extended use of reagent cocktails. In this study, we present a simplified method to efficiently generate large numbers of uniformly differentiated keratinocytes in less than 4 weeks from singularized hiPSCs with differentiation factors, retinoic acid and bone morphogenetic protein 4 (BMP4). Low seeding density of singularized iPSCs results in keratinocyte cultures with minimum cell death during differentiation and up to 96% homogeneity for keratin 14-positive cells and low percentage of keratinocyte maturation markers, comparable to early passage primary keratinocytes. hiPSC-derived keratinocytes remain in a proliferative state, can be maintained for prolonged periods of time, and can be terminally differentiated under high calcium conditions in the same way as primary human keratinocytes. Moreover, coculturing hiPSC-derived fibroblasts and keratinocytes consistently formed organotypic 3D skin equivalents. Therefore, keratinocytes generated by this method are a viable source of cells for downstream applications.
| S-EPMC8665820 | biostudies-literature
Project description:Human pluripotent stem cells hold great promise for their practical and scientific potentials. To improve understanding of self-renewal and differentiation, we previously reported a defined serum-free medium hESF9 could generate and maintain human induced pluripotent stem cells (iPSCs) in serum- and feeder-free culture conditions using retroviral vectors. To avoid the unpredictable side effects associated with retrovirus integration, we report here the successful generation of hiPSCs from dental pulp cells with a non-integrating replication-defective and persistent Sendai virus (SeVdp) vector expressing four key reprogramming genes. We found that hESF9 medium in combination with fibronectin are effective for generating and maintaining hiPSCs with SeVdp (KOSM). Using this system, pluripotent and self-renewing hiPSCs could be easily and stably generated and propagated. With this system, we successfully generated hiPSCs from cleidocranial dysplasia (CCD) caused by a heterozygous germ-line mutation of runt-related protein2 (RUNX2), which has an important role in the differentiation of osteoblasts and maturation of chondrocytes. This is the first report of the establishment of CCD-specific iPSCs. The cartilage in the teratomas of CCD-iPSCs showed abnormalities. These CCD-iPSCs would be beneficial to clarify the molecular mechanism and for development of medical applications. Moreover, it brings new pathophysiological role of RUNX2 in the differentiation of the human chondrocytes and osteocytes.
| S-EPMC4746228 | biostudies-literature
Project description:Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC) to maintain their pluripotent self-renewal capacity during in vitro culture. hPSCs secrete laminin 511/521, one of the most important functional basement membrane components, and they can be maintained on human laminin 511 and 521 in defined culture conditions. However, large-scale production of purified or recombinant laminin 511 and 521 is difficult and expensive. Here we have tested whether a commonly available human choriocarcinoma cell line, JAR, which produces high quantities of laminins, supports the growth of undifferentiated hPSCs. We were able to maintain several human pluripotent stem cell lines on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly, we were able to derive new human induced pluripotent stem cell (hiPSC) lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary, JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture and differentiation. In addition, this matrix is ideal for the efficient generation of new hiPSC lines.
| S-EPMC3788803 | biostudies-literature
Project description:The pluripotency maintenance of pluripotent stem cells (PSCs) requires the suitable microenvironment, which commonly provided by feeder layers. However, the preparation of feeder layers is time consuming and labor exhaustive, and the feeder cells treated with mitomycin C or γ-ray irradiation bring heterologous contamination. In this study, mouse embryonic fibroblasts (MEFs) were treated by methanol to generate chemical fixed feeder cells, and bovine embryonic stem cells F7 (bESC-F7) cultured on this feeder layer. Then the pluripotency and metabolism of bESC-F7 cultured on methanol-fixed MEFs (MT-MEFs) named MT-F7 was compared with mitomycin C treated MEFs (MC-MEFs). The results showed that bESC-F7 formed alkaline phosphatase positive colonies on MT-MEFs, the relative expression of pluripotent markers of these cells was different from the bESCs cultured on the MC-MEFs (MC-F7). The long-term cultured MT-F7 formed embryoid bodies, showed the ability to differentiate into three germ layers similar to MC-F7. The analyses of RNA-seq data showed that MT-MEFs lead bESCs to novel steady expression patterns of genes regulating pluripotency and metabolism. Furthermore, the bovine expanded pluripotent stem cells (bEPSCs) cultured on MT-MEFs formed classical colonies, maintained pluripotency, and elevated metabolism. In conclusion, MT-MEFs were efficient feeder layer that maintain the distinctive pluripotency and metabolism of PSCs.
| S-EPMC9163156 | biostudies-literature
Project description:Genome integration-free pig induced pluripotent stem cells (iPSCs) bring tremendous value in pre-clinical testing of regenerative medicine, as well as conservation and exploitation of endangered or rare local pig idioplasmatic resources. However, due to a lack of appropriate culture medium, efficient induction and stable maintenance of pig iPSCs with practical value remains challenging. Here, we established an efficient induction system for exogenous gene-independent iPSCs under chemically defined culture condition previously used for generation of stable pig pre-gastrulation epiblast stem cells (pgEpiSCs). WNT suppression was found to play an essential role in establishment of exogenous gene-independent iPSCs. Strikingly, stable integration-free pig iPSCs could be established from pig somatic cells using episomal vectors in this culture condition. The iPSCs had pluripotency features and transcriptome characteristics approximating pgEpiSCs. More importantly, this induction system may be used to generate integration-free iPSCs from elderly disabled rare local pig somatic cells and the iPSCs could be gene-edited and used as donor cells for nuclear transfer. Our results provide novel insights into potential applications for genetic breeding of livestock species and pre-clinical evaluation of regenerative medicine.
| S-EPMC10623960 | biostudies-literature
Project description:Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.
| S-EPMC8197809 | biostudies-literature
Project description:Human ES cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are usually generated and maintained on living feeder cells like mouse embryonic fibroblasts or on a cell-free substrate like Matrigel. For clinical applications, a quality-controlled, xenobiotic-free culture system is required to minimize risks from contaminating animal-derived pathogens and immunogens. We previously reported that the pericellular matrix of decidua-derived mesenchymal cells (PCM-DM) is an ideal human-derived substrate on which to maintain hiPSCs/hESCs. In this study, we examined whether PCM-DM could be used for the generation and long-term stable maintenance of hiPSCs. Decidua-derived mesenchymal cells (DMCs) were reprogrammed by the retroviral transduction of four factors (OCT4, SOX2, KLF4, c-MYC) and cultured on PCM-DM. The established hiPSC clones expressed alkaline phosphatase, hESC-specific genes and cell-surface markers, and differentiated into three germ layers in vitro and in vivo. At over 20 passages, the hiPSCs cultured on PCM-DM held the same cellular properties with genome integrity as those at early passages. Global gene expression analysis showed that the GDF3, FGF4, UTF1, and XIST expression levels varied during culture, and GATA6 was highly expressed under our culture conditions; however, these gene expressions did not affect the cells' pluripotency. PCM-DM can be conveniently prepared from DMCs, which have a high proliferative potential. Our findings indicate that PCM-DM is a versatile and practical human-derived substrate that can be used for the feeder-cell-free generation and long-term stable maintenance of hiPSCs.
| S-EPMC3561375 | biostudies-literature
Project description:The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.
| S-EPMC5841662 | biostudies-literature