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Enabling Large-Scale Ex Vivo Production of Megakaryocytes from CD34⁺ Cells Using Gas-Permeable Surfaces.

ABSTRACT: Patients suffering from acute or sustained thrombocytopenia require platelet transfusions, which are entirely donor-based and limited by challenges related to storage and fluctuating supply. Developing cell-culture technologies will enable ex vivo and donor-independent platelet production. However, critical advancements are needed to improve scalability and increase megakaryocyte (Mk) culture productivity. To address these needs, we evaluated Mk production from mobilized peripheral blood CD34⁺ cells cultured on a commercially available gas-permeable silicone rubber membrane, which provides efficient gas exchange, and investigated the use of fed-batch media dilution schemes. Starting with a cell-surface density of 40 × 10³ CD34⁺ cells per cm² (G40D), culturing cells on the membrane for the first 5 days and employing media dilutions yielded 39?±?19 CD41⁺ CD42b⁺ Mks per input CD34⁺ cell by day 11-a 2.2-fold increase compared with using standard culture surfaces and full media exchanges. By day 7, G40D conditions generated 1.5-fold more CD34⁺ cells and nearly doubled the numbers of Mk progenitors. The increased number of Mk progenitors coupled with media dilutions, potentially due to the retention of interleukin (IL)-3, increased Mk production in G40D. Compared with controls, G40D had higher viability, yielded threefold more Mks per milliliter of media used and exhibited lower mean ploidy, but had higher numbers of high-ploidy Mks. Finally, G40D-Mks produced proplatelets and platelet-like-particles that activate and aggregate upon stimulation. These results highlight distinct improvements in Mk cell-culture and demonstrate how new technologies and techniques are needed to enable clinically relevant production of Mks for platelet generation and cell-based therapies.

SUBMITTER: Martinez AF

PROVIDER: S-EPMC6591548 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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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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Enabling Large-Scale Ex Vivo Production of Megakaryocytes from CD34⁺ Cells Using Gas-Permeable Surfaces.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Enabling Large-Scale Ex Vivo Production of Megakaryocytes from CD34+ Cells Using Gas-Permeable Surfaces.

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OmicsDI is part of the ELIXIR infrastructure

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Enabling Large-Scale Ex Vivo Production of Megakaryocytes from CD34⁺ Cells Using Gas-Permeable Surfaces.