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Recapitulative hematopoietic development of human pluripotent stem cells in the absence of exogenous cytokines

ABSTRACT: We tested whether the sustained hematopoietic differentiation of hPSCs can be achieved in defined cell culture conditions without addition of hematopoietic cytokines. Here we show that the endogenous stimuli were sufficient to induce a robust generation of clonogenic hematopoietic progenitors, maturation of blood cells, emergence of the definitive cell lineages, and progenitors that were phenotypically identical to early human embryonic hematopoietic stem cells (HSCs). Our novel cytokine-free protocol is efficient, reproducible, and can be applied for hematopoietic differentiation of human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs). The removal of cytokines from the differentiation led to detecting a vast developmental potential of the early human blood cells. We also found that the cytokine-free hematopoietic differentiation of hPSCs is associated with strong activation of hPSC-derived inflammatory cells. The spectrum of recapitulative features of the novel protocol makes the cytokine-free differentiation a preferred model for studying early human hematopoietic development

ORGANISM(S): Homo sapiens

PROVIDER: GSE159672 | GEO | 2020/10/21

REPOSITORIES: GEO

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Project description:Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many of the potential applications are still limited by the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. These challenges could be overcome by the use of adult tissue stem cells derived from hPSCs, as their restricted potential could limit the differentiation towards other undesired linages, and allow in vitro expansion and long- term propagation of fully differentiated tissue. To isolate adult stem cells from hPSCs, we applied genome-editing to generate an LGR5-GFP reporter system and subsequently developed a differentiation protocol for human intestinal tissue comprising an adult stem cell niche and all major cell types of the adult intestine. This novel derivation protocol is highly robust and even permits the isolation of intestinal organoids without the LGR5 reporter. Transcriptional profiling, electron microscopy and functional analysis revealed that such human organoid cultures could be derived with high purity, and a composition and morphology similar to that of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. With our ability to genetically engineer hPSCs using site-specific nucleases, this adult stem cell system provides a novel platform by which to study human intestinal disease in vitro. RNA from primary organoid samples was isolated from organoid lines that were both cultured for 1-6 months and derived from duodenum, ileum, or rectum biopsies of human subjects as described previously (Sato et al., Gastroenterology 2011) grown in media called WENR+inhibitors. RNA was also isolated from various steps in the culturing and differentiation protocol.

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Recapitulative hematopoietic development of human pluripotent stem cells in the absence of exogenous cytokines

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