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Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells.

ABSTRACT: Understanding physiologic T-cell development from hematopoietic stem (HSCs) and progenitor cells (HPCs) is essential for development of improved hematopoietic cell transplantation (HCT) and emerging T-cell therapies. Factors in the thymic niche, including Notch 1 receptor ligand, guide HSCs and HPCs through T-cell development in vitro. We report that physiologically relevant oxygen concentration (5% O₂ , physioxia), an important environmental thymic factor, promotes differentiation of cord blood CD34+ cells into progenitor T (proT) cells in serum-free and feeder-free culture system. This effect is enhanced by a potent reducing and antioxidant agent, ascorbic acid. Human CD34+ cell-derived proT cells in suspension cultures maturate into CD3+ T cells in an artificial thymic organoid (ATO) culture system more efficiently when maintained under physioxia, compared to ambient air. Low oxygen tension acts as a positive regulator of HSC commitment and HPC differentiation toward proT cells in the feeder-free culture system and for further maturation into T cells in the ATO. Culturing HSCs/HPCs in physioxia is an enhanced method of effective progenitor T and mature T-cell production ex vivo and may be of future use for HCT and T-cell immunotherapies.

SUBMITTER: Shin DY

PROVIDER: S-EPMC7901063 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells.

Shin Dong-Yeop DY Huang Xinxin X Gil Chang-Hyun CH Aljoufi Arafat A Ropa James J Broxmeyer Hal E HE

Stem cells (Dayton, Ohio) 20200815 11

Understanding physiologic T-cell development from hematopoietic stem (HSCs) and progenitor cells (HPCs) is essential for development of improved hematopoietic cell transplantation (HCT) and emerging T-cell therapies. Factors in the thymic niche, including Notch 1 receptor ligand, guide HSCs and HPCs through T-cell development in vitro. We report that physiologically relevant oxygen concentration (5% O<sub>2</sub> , physioxia), an important environmental thymic factor, promotes differentiation of ...[more]

PMID: 32761664

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Project description:Despite evidence that SARS-CoV-2 infection is systemic in nature, there is little known about the effects that SARS-CoV-2 infection or exposure has on many host cell types, including primitive and mature hematopoietic cells. The hematopoietic system is responsible for giving rise to the very immune cells that defend against viral infection and is a source of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) which are used for hematopoietic cell transplantation (HCT) to treat hematologic disorders, thus there is a strong need to understand how exposure to the virus may affect hematopoietic cell functions. We examined the expression of ACE2, to which SARS-CoV-2 Spike (S) protein binds to facilitate viral entry, in cord blood derived HSCs/HPCs and in peripheral blood derived immune cell subtypes. ACE2 is expressed in low numbers of immune cells, higher numbers of HPCs, and up to 65% of rigorously defined HSCs. We also examined effects of exposing HSCs/HPCs and immune cells to SARS-CoV-2 S protein ex vivo. HSCs and HPCs expand less effectively and have less functional colony forming capacity when grown with S protein, while peripheral blood monocytes upregulate CD14 expression and show distinct changes in size and granularity. That these effects are induced by recombinant S protein alone and not the infectious viral particle suggests that simple exposure to SARS-CoV-2 may impact HSCs/HPCs and immune cells via S protein interactions with the cells, regardless of whether they can be infected. These data have implications for immune response to SARS-CoV-2 and for HCT. Graphical Abstract • Human HSCs, HPCs, and immune cells express ACE2 on the cell surface, making them potentially susceptible to SARS-CoV-2 infection. • SARS-CoV-2 S protein, which binds to ACE2, induces defects in the colony forming capacity of human HPC and inhibits the expansion of HSC/HPC subpopulations ex vivo. These effects can be at least partially neutralized by treatment with SARS-CoV-2 targeting antibody, recombinant human ACE2, or Angiotensin1-7. • S protein also induces aberrant morphological changes in peripheral blood derived monocytes ex vivo. • Thus, there are many different manners in which SARS-CoV-2 virus may impact the functional hematopoietic system, which has important implications for hematological manifestations of COVID-19 (i.e. thrombocytopenia and lymphopenia), immune response, and hematopoietic stem cell transplant in the era of COVID-19.

Project description:BackgroundMesenchymal stem/stromal cells (MSCs) are of great therapeutic value due to their role in maintaining the function of hematopoietic stem/progenitor cells (HSPCs). MSCs derived from human pluripotent stem cells represent an ideal alternative because of their unlimited supply. However, the role of MSCs with neural crest origin derived from HPSCs on the maintenance of HSPCs has not been reported.MethodsFlow cytometric analysis, RNA sequencing and differentiation ability were applied to detect the characteristics of stromal cells from 3D human brain organoids. Human umbilical cord blood CD34+ (UCB-CD34+) cells were cultured in different coculture conditions composed of stromal cells and umbilical cord MSCs (UC-MSCs) with or without a cytokine cocktail. The hematopoietic stroma capacity of stromal cells was tested in vitro with the LTC-IC assay and in vivo by cotransplantation of cord blood nucleated cells and stroma cells into immunodeficient mice. RNA and proteomic sequencing were used to detect the role of MSCs on HSPCs.ResultsThe stromal cells, derived from both H1-hESCs and human induced pluripotent stem cells forebrain organoids, were capable of differentiating into the classical mesenchymal-derived cells (osteoblasts, chondrocytes, and adipocytes). These cells expressed MSC markers, thus named pluripotent stem cell-derived MSCs (pMSCs). The pMSCs showed neural crest origin with CD271 expression in the early stage. When human UCB-CD34+ HSPCs were cocultured on UC-MSCs or pMSCs, the latter resulted in robust expansion of UCB-CD34+ HSPCs in long-term culture and efficient maintenance of their transplantability. Comparison by RNA sequencing indicated that coculture of human UCB-CD34+ HSPCs with pMSCs provided an improved microenvironment for HSC maintenance. The pMSCs highly expressed the Wnt signaling inhibitors SFRP1 and SFRP2, indicating that they may help to modulate the cell cycle to promote the maintenance of UCB-CD34+ HSPCs by antagonizing Wnt activation.ConclusionsA novel method for harvesting MSCs with neural crest origin from 3D human brain organoids under serum-free culture conditions was reported. We demonstrate that the pMSCs support human UCB-HSPC expansion in vitro in a long-term culture and the maintenance of their transplantable ability. RNA and proteomic sequencing indicated that pMSCs provided an improved microenvironment for HSC maintenance via mechanisms involving cell-cell contact and secreted factors and suppression of Wnt signaling. This represents a novel method for large-scale production of MSCs of neural crest origin and provides a potential approach for development of human hematopoietic stromal cell therapy for treatment of dyshematopoiesis.

Project description:Umbilical cord blood (UCB) has been established as an alternative source for hematopoietic stem/progenitor cells (HSPC) for cell and gene therapies. Limited cell yields of UCB units have been tackled with the development of cytokine-based ex vivo expansion platforms. To improve the effectiveness of these platforms, namely targeting clinical approval, in this study, we optimized the cytokine cocktails in two clinically relevant expansion platforms for HSPC, a liquid suspension culture system (CS_HSPC) and a co-culture system with bone marrow derived mesenchymal stromal cells (BM MSC) (CS_HSPC/MSC). Using a methodology based on experimental design, three different cytokines [stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin (TPO)] were studied in both systems during a 7-day culture under serum-free conditions. Proliferation and colony-forming unit assays, as well as immunophenotypic analysis were performed. Five experimental outputs [fold increase (FI) of total nucleated cells (FI TNC), FI of CD34+ cells, FI of erythroid burst-forming unit (BFU-E), FI of colony-forming unit granulocyte-monocyte (CFU-GM), and FI of multilineage colony-forming unit (CFU-Mix)] were followed as target outputs of the optimization model. The novel optimized cocktails determined herein comprised concentrations of 64, 61, and 80 ng/mL (CS_HSPC) and 90, 82, and 77 ng/mL (CS_HSPC/MSC) for SCF, Flt-3L, and TPO, respectively. After cytokine optimization, CS_HSPC and CS_HSPC/MSC were directly compared as platforms. CS_HSPC/MSC outperformed the feeder-free system in 6 of 8 tested experimental measures, displaying superior capability toward increasing the number of hematopoietic cells while maintaining the expression of HSPC markers (i.e., CD34+ and CD34+CD90+) and multilineage differentiation potential. A tailored approach toward optimization has made it possible to individually maximize cytokine contribution in both studied platforms. Consequently, cocktail optimization has successfully led to an increase in the expansion platform performance, while allowing a rational side-by-side comparison among different platforms and enhancing our knowledge on the impact of cytokine supplementation on the HSPC expansion process.

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Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells.

Publications

Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells.

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