Direct lineage reprogramming, including with small molecules, has emerged as a promising approach for generating desired cell types. We recently found that during chemical induction of induced ...pluripotent stem cells (iPSCs) from mouse fibroblasts, cells pass through an extra-embryonic endoderm (XEN)-like state. Here, we show that these chemically induced XEN-like cells can also be induced to directly reprogram into functional neurons, bypassing the pluripotent state. The induced neurons possess neuron-specific expression profiles, form functional synapses in culture, and further mature after transplantation into the adult mouse brain. Using similar principles, we were also able to induce hepatocyte-like cells from the XEN-like cells. Cells in the induced XEN-like state were readily expandable over at least 20 passages and retained genome stability and lineage specification potential. Our study therefore establishes a multifunctional route for chemical lineage reprogramming and may provide a platform for generating a diverse range of cell types via application of this expandable XEN-like state.
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•Chemically induced XEN-like state is plastic to be induced into functional cell types•Functional neurons and hepatocytes are induced from the XEN-like state•XEN-like cells maintain genome stability and properties during long-term expansion
In this article, Deng and colleagues show that functional neurons and hepatocytes can be induced from fibroblasts via a chemically induced and highly expandable XEN-like state, bypassing the pluripotent stage. This featured lineage reprogramming strategy would be generalized for generating other functional desirable cell types.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Extended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free ...culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.
Terminally differentiated cells can be generated by lineage reprogramming, which is, however, hindered by incomplete conversion with residual initial cell identity and partial functionality. Here, we ...demonstrate a new reprogramming strategy by mimicking the natural regeneration route, which permits generating expandable hepatic progenitor cells and functionally competent human hepatocytes. Fibroblasts were first induced into human hepatic progenitor-like cells (hHPLCs), which could robustly expand in vitro and efficiently engraft in vivo. Moreover, hHPLCs could be efficiently induced into mature human hepatocytes (hiHeps) in vitro, whose molecular identity highly resembles primary human hepatocytes (PHHs). Most importantly, hiHeps could be generated in large quantity and were functionally competent to replace PHHs for drug-metabolism estimation, toxicity prediction and hepatitis B virus infection modeling. Our results highlight the advantages of the progenitor stage for successful lineage reprogramming. This strategy is promising for generating other mature human cell types by lineage reprogramming.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult ...organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research.
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•A chemical cocktail enables derivation of EPS cells from both humans and mice•A single mouse EPS cell generates both embryonic and extraembryonic lineages in vivo•Mouse EPS cells show robust and superior chimeric ability at the single cell level•Human EPS cells show interspecies chimeric competency in mouse conceptuses
Pluripotent stem cells in a defined chemical culture condition can generate both embryonic and extraembryonic tissues at the single-cell level.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cellular reprogramming can manipulate the identity of cells to generate the desired cell types
. The use of cell intrinsic components, including oocyte cytoplasm and transcription factors, can ...enforce somatic cell reprogramming to pluripotent stem cells
. By contrast, chemical stimulation by exposure to small molecules offers an alternative approach that can manipulate cell fate in a simple and highly controllable manner
. However, human somatic cells are refractory to chemical stimulation owing to their stable epigenome
and reduced plasticity
; it is therefore challenging to induce human pluripotent stem cells by chemical reprogramming. Here we demonstrate, by creating an intermediate plastic state, the chemical reprogramming of human somatic cells to human chemically induced pluripotent stem cells that exhibit key features of embryonic stem cells. The whole chemical reprogramming trajectory analysis delineated the induction of the intermediate plastic state at the early stage, during which chemical-induced dedifferentiation occurred, and this process was similar to the dedifferentiation process that occurs in axolotl limb regeneration. Moreover, we identified the JNK pathway as a major barrier to chemical reprogramming, the inhibition of which was indispensable for inducing cell plasticity and a regeneration-like program by suppressing pro-inflammatory pathways. Our chemical approach provides a platform for the generation and application of human pluripotent stem cells in biomedicine. This study lays foundations for developing regenerative therapeutic strategies that use well-defined chemicals to change cell fates in humans.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Transplantation of hematopoietic stem cells (HSCs) with a naturally occurring CCR5 mutation confers a loss of detectable HIV-1 in the patient, making ablation of the CCR5 gene in HSCs an ideal ...therapy for an HIV-1 cure. Although CCR5 disruption has been attempted in CD4+ T cells and hematopoietic stem/progenitor cells (HSPCs), efficient gene editing with high specificity and long-term therapeutic potential remains a major challenge for clinical translation. Here, we established a CRISPR/Cas9 gene editing system in human CD34+ HSPCs and achieved efficient CCR5 ablation evaluated in long-term reconstituted NOD/Prkdcscid/IL-2Rγnull mice. The CCR5 disruption efficiency in our system remained robust in secondary transplanted repopulating hematopoietic cells. More importantly, an HIV-1 resistance effect was observed as indicated by significant reduction of virus titration and enrichment of human CD4+ T cells. Hence, we successfully established a CRISPR/Cas9 mediated CCR5 ablating system in long-term HSCs, which confers HIV-1 resistance in vivo. Our study provides evidence for translating CCR5 gene-edited HSC transplantation for an HIV cure to the clinic.
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Xu et al. have used the CRISPR/Cas9 system to achieve CCR5 gene disruption in human hematopoietic stem/progenitor cells (HSPCs). The gene-ablated HSPCs could reconstitute in vivo long term and resist HIV-1 infection. This study provides evidence of CCR5 gene ablation in HSCs as a promising therapy for HIV-1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
RNA polymerase II (RNA Pol II) subunits are thought to be involved in various transcription-associated processes, but it is unclear whether they play different regulatory roles in modulating gene ...expression. Here, we performed nascent and mature transcript sequencing after the acute degradation of 12 mammalian RNA Pol II subunits and profiled their genomic binding sites and protein interactomes to dissect their molecular functions. We found that RNA Pol II subunits contribute differently to RNA Pol II cellular localization and transcription processes and preferentially regulate RNA processing (such as RNA splicing and 3′ end maturation). Genes sensitive to the depletion of different RNA Pol II subunits tend to be involved in diverse biological functions and show different RNA half-lives. Sequences, associated protein factors, and RNA structures are correlated with RNA Pol II subunit-mediated differential gene expression. These findings collectively suggest that the heterogeneity of RNA Pol II and different genes appear to depend on some of the subunits.
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•Twelve mammalian RNA Pol II subunits are functionally characterized•Loss of RPB3, RPB9, RPB10, and RPB11 affects the expression of a subset of genes•RNA Pol II subunits contribute differentially to gene regulation•RNA Pol II is an optimized polymerase involved in diverse processes through its subunits
Li and Huang et al. show that RNA Pol II subunits are heterogeneous, and different genes are preferentially regulated by certain subunits in mammalian cells. This indicates that acute protein degradation combined with multiomic analyses promise insights into other unexplored subunits in multimolecular complexes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Eosinophils are attractive innate immune cells to use to potentiate T cell antitumor efficacy because they are capable of infiltrating tumors at early stages and modulating the tumor ...microenvironment. However, the limited number of functional eosinophils caused by the scarcity and short life of primary eosinophils in peripheral blood has greatly impeded the development of eosinophil-based immunotherapy. In this study, we established an efficient chemically defined protocol to generate a large quantity of functional eosinophils from human pluripotent stem cells (hPSCs) with nearly 100% purity expressing eosinophil peroxidase. These hPSC-derived eosinophils transcriptionally resembled their primary counterpart. Moreover, hPSC-derived eosinophils showed competent tumor killing capacity in established solid tumors. Furthermore, the combination of hPSC-derived eosinophils with CAR-T cells exhibited potential synergistic effects, inhibiting tumor growth and enhancing mouse survival. Our study opens up new avenues for the development of eosinophil-based immunotherapies to treat cancer.
•A chemically defined approach generates functional human eosinophils from hPSCs•hPSC-derived eosinophils exhibit competent antitumor ability in vitro and in vivo•Potential synergy of action when both eosinophils and CAR-T cells are used together
In this article, Deng and colleagues establish an efficient chemically defined protocol to generate functional eosinophils from hPSCs, which show competent tumor killing capacity in vitro and in vivo. In addition, the combination of hPSC-derived eosinophils with CAR-T cells exhibits potential synergistic effects suppressing tumor growth. This study opens up a new avenue for developing eosinophil-based immunotherapies against solid tumors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The limited number of human hematopoietic stem cells (HSCs) has restrained their widespread clinical application. Despite great efforts in recent years, the in vitro expansion of HSCs remains a ...challenge due to incomplete understanding of the signaling networks underlying HSC self-renewal. Here, we show that culturing human cord blood (CB) CD34
cells with JNK-IN-8, an inhibitor of the JNK signaling pathway, can enhance the self-renewal of HSCs with a 3.88-fold increase in cell number. These cultured CD34
cells repopulated recipient mice for 21 weeks and can form secondary engraftment that lasted for more than 21 weeks. Knockdown of
, a major downstream target in the JNK pathway, promoted the expansion of hematopoietic stem and progenitor cells (HSPCs). Our findings demonstrate a critical role of the JNK pathway in regulating HSC expansion, provide new insights into HSC self-renewal mechanism, and may lead to improved clinical application of HSCs.
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