Feeder-Free Derivation of Naive Human Pluripotent Stem Cells.
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ABSTRACT:
SUBMITTER: Ward E
PROVIDER: S-EPMC5563919 | biostudies-literature | 2017 Aug
REPOSITORIES: biostudies-literature
Publications
Feeder-Free Derivation of Naive Human Pluripotent Stem Cells.
Ward Emily E Twaroski Kirk K Tolar Jakub J
Stem cells and development 20170630 15
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Project description:Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.
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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.
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Project description:BackgroundHuman pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (PSCs) have the capacity of self-renewal and multilineage differentiation in vitro. Conventional hPSCs, which are in a primed state, can produce various types of differentiated cells. However, the variability in their degree of pluripotency and differentiation propensities, which is influenced by the inductive methods and culture conditions, limit their availability. Therefore, PSCs in a naïve state are a promising source of PSCs.MethodsWe recently developed a culture system for naïve hPSCs using an inhibitor of the NOTCH signaling pathway and a histone H3 methyltransferase disruptor. This culture system requires feeder cells for stably maintaining the naïve hPSCs. We aimed to develop a culture system for hPSCs that could maintain pluripotency under feeder-free conditions.ResultsWe used two inhibitors to develop an alternative feeder-free culture system to obtain naïve hPSCs. The naïve cells underwent stable cellular proliferation and were positive for naïve stem cell markers; in addition, they could differentiate into the three germ layers. These feeder-free dome-shaped induced pluripotent stem cells (FFDS-iPSCs) have characteristics similar to that of naïve-like PSCs.ConclusionsThe naive hPSCs under feeder-free conditions could ensure supply of cells for various applications in regenerative medicine and disease modeling.
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Project description:Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.
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