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Limited Mitochondrial Activity Coupled With Strong Expression of CD34, CD90 and EPCR Determines the Functional Fitness of ex vivo Expanded Human Hematopoietic Stem Cells.

ABSTRACT: Ex vivo expansion strategies of human hematopoietic stem cell (HSC) grafts with suboptimal stem cell dose have emerged as promising strategies for improving outcomes of HSC transplantation in patients with hematological malignancies. While exposure of HSCs to ex vivo cultures expands the number of phenotypically identifiable HSCs, it frequently alters the transcriptomic and metabolic profiles, therefore, compromising their long-term (LT) hematopoietic reconstitution capacity. Within the heterogeneous pool of expanded HSCs, the precise phenotypic, transcriptomic and metabolic profile and thus, the identity of HSCs that confer LT repopulation potential remains poorly described. Utilizing valproic acid (VPA) in ex vivo cultures of umbilical cord blood (UCB)-CD34⁺ cells, we demonstrate that expanded HSCs phenotypically marked by expression of the stem cell markers CD34, CD90 and EPCR (CD201) are highly enriched for LT-HSCs. Furthermore, we report that low mitochondrial membrane potential, and, hence, mitochondrial activity distinguishes LT-HSCs within the expanded pool of phenotypically defined HSCs. Remarkably, such reduced mitochondrial activity is restricted to cells with the highest expression levels of CD34, CD90 and EPCR phenotypic markers. Together, our findings reveal that high expression of CD34, CD90 and EPCR in conjunction with low mitochondrial activity is critical for identification of functional LT-HSCs generated within ex vivo expansion cultures.

SUBMITTER: Papa L

PROVIDER: S-EPMC7769876 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Limited Mitochondrial Activity Coupled With Strong Expression of CD34, CD90 and EPCR Determines the Functional Fitness of ex vivo Expanded Human Hematopoietic Stem Cells.

Papa Luena L Djedaini Mansour M Martin Tiphaine C TC Zangui Mahtab M Beaumont Kristin G KG Sebra Robert R Parsons Ramon R Schaniel Christoph C Hoffman Ronald R

Frontiers in cell and developmental biology 20201215

Ex vivo expansion strategies of human hematopoietic stem cell (HSC) grafts with suboptimal stem cell dose have emerged as promising strategies for improving outcomes of HSC transplantation in patients with hematological malignancies. While exposure of HSCs to ex vivo cultures expands the number of phenotypically identifiable HSCs, it frequently alters the transcriptomic and metabolic profiles, therefore, compromising their long-term (LT) hematopoietic reconstitution capacity. Withi ...[more]

PMID: 33384995

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Project description:Diabetes can impair wound closure, which can give rise to major clinical problems. Most treatments for wound repair in diabetes remain ineffective. This study aimed to investigate the influence on wound closure of treatments using expanded human cord blood CD34(+) cells (CB-CD34(+) cells), freshly isolated CB-CD34(+) cells and a cytokine cocktail. The test subjects were mice with streptozotocin-induced diabetes. Wounds treated with fresh CB-CD34(+) cells showed more rapid repair than mice given the PBS control. Injection of expanded CB-CD34(+) cells improved wound closure significantly, whereas the injection of the cytokine cocktail alone did not improve wound repair. The results also demonstrated a significant decrease in epithelial gaps and advanced re-epithelialization over the wound bed area after treatment with either expanded CB-CD34(+) cells or freshly isolated cells compared with the control. In addition, treatments with both CB-CD34(+) cells and the cytokine cocktail were shown to promote recruitment of CD31(+)-endothelial cells in the wounds. Both the CB-CD34(+) cell population and the cytokine treatments also enhanced the recruitment of CD68-positive cells in the early stages (day 3) of treatment compared with PBS control, although the degree of this enhancement was found to decline in the later stages (day 9). These results demonstrated that expanded CB-CD34(+) cells or freshly isolated CB-CD34(+) cells could accelerate wound repair by increasing the recruitment of macrophages and capillaries and the reepithelialization over the wound bed area. Our data suggest an effective role in wound closure for both ex vivo expanded CB-CD34(+) cells and freshly isolated cells, and these may serve as therapeutic options for wound treatment for diabetic patients. Wound closure acceleration by expanded CB-CD34(+) cells also breaks the insufficient quantity obstacle of stem cells per unit of cord blood and other stem cell sources, which indicates a broader potential for autologous transplantation.

Project description:BackgroundHematopoietic stem cell (HSC) transplantation has been widely applied to the treatment of malignant blood diseases. However, limited number of functional HSCs hinders successful transplantation. The purpose of our current study is to develop a new and cost-efficient medium formulation that could greatly enhance the expansion of HSCs while retaining their long-term repopulation and hematopoietic properties for effective clinical transplantation.MethodsEnriched human CD34+ cells and mobilized nonhuman primate peripheral blood CD34+ cells were expanded with a new, cost-efficient expansion medium formulation, named hematopoietic expansion medium (HEM), consisting of various cytokines and nutritional supplements. The long-term repopulation potential and hematologic-lineage differentiation ability of expanded human cells were studied in the non-obese diabetic/severe combined immunodeficiency mouse model. Furthermore, the efficacy and safety studies were performed by autologous transplantation of expanded primate cells in the nonhuman primate model.ResultsHEM could effectively expand human CD34+ cells by up to 129 fold within 9 days. Expanded HSCs retained long-term repopulation potential and hematologic-lineage differentiation ability, as indicated by (1) maintenance (over unexpanded HSCs) of immunophenotypes of CD38-CD90+CD45RA-CD49f+ in CD34+ cells after expansion; (2) significant presence of multiple human hematopoietic lineages in mouse peripheral blood and bone marrow following primary transplantation; (3) enrichment (over unexpanded HSCs) in SCID-repopulating cell frequency measured by limiting dilution analysis; and (4) preservation of both myeloid and lymphoid potential among human leukocytes from mouse bone marrow in week 24 after primary transplantation or secondary transplantation. Moreover, the results of autologous transplantation in nonhuman primates demonstrated that HEM-expanded CD34+ cells could enhance hematological recovery after myelo-suppression. All primates transplanted with the expanded autologous CD34+ cells survived for over 18 months without any noticeable abnormalities.ConclusionsTogether, these findings demonstrate promising potential for the utility of HEM to improve expansion of HSCs for clinical application.

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Limited Mitochondrial Activity Coupled With Strong Expression of CD34, CD90 and EPCR Determines the Functional Fitness of ex vivo Expanded Human Hematopoietic Stem Cells.

Publications

Limited Mitochondrial Activity Coupled With Strong Expression of CD34, CD90 and EPCR Determines the Functional Fitness of <i>ex vivo</i> Expanded Human Hematopoietic Stem Cells.

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