Cytotherapy · 2026
Uncovering molecular and functional dynamics of human hematopoietic stem cells during in vitro culture for cell therapy manufacturing.
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Xavier-Ferrucio Juliana, Ung Matthew, Norman Fulton, Li Bin E, Manalo Nate, Pechilis Lisa, Ge Gary, Chakraborty Tirtha, Lin Michelle I
Аннотация
BACKGROUND: Background: Hematopoietic stem/progenitor cell (HSPC) transplantation outcomes depend critically on the dose of functional stem cells delivered to patients, yet current ex vivo cell therapy manufacturing processes lack reliable methods to quantify therapeutic hematopoietic stem cells (HSCs) after in vitro culture. This gap poses significant clinical risks, as culture-induced changes may alter the cellular composition of therapeutic products. The challenge stems from two key limitations: (i) biological-conventional HSC markers to phenotypically characterize subpopulations such as CD38 become unreliable during culture, and (ii) technical-existing assays cannot distinguish between HSCs and phenotypically similar progenitors in manipulated products.
AIM: To solve this problem, we compared an ITGA3- and CD49f-based identification strategy that bypasses culture-sensitive markers.
METHODS: Through single-cell analysis of adult mobilized peripheral blood HSPCs cultured under clinical manufacturing conditions, we demonstrate that ITGA3+ cells better maintain stemness signatures (higher AVP/HLF expression) compared to CD49f+.
RESULTS: Functional validation through xenotransplantation revealed superior engraftment and multilineage reconstitution with ITGA3+ HSCs.
CONCLUSIONS: Our findings provide the first validated method to track functional HSCs during clinical manufacturing, addressing a fundamental quality control challenge in the field. This advance enables more accurate dosing for transplants and provides a critical tool for emerging gene-modified HSPC therapies where product characterization is paramount.
AIM: To solve this problem, we compared an ITGA3- and CD49f-based identification strategy that bypasses culture-sensitive markers.
METHODS: Through single-cell analysis of adult mobilized peripheral blood HSPCs cultured under clinical manufacturing conditions, we demonstrate that ITGA3+ cells better maintain stemness signatures (higher AVP/HLF expression) compared to CD49f+.
RESULTS: Functional validation through xenotransplantation revealed superior engraftment and multilineage reconstitution with ITGA3+ HSCs.
CONCLUSIONS: Our findings provide the first validated method to track functional HSCs during clinical manufacturing, addressing a fundamental quality control challenge in the field. This advance enables more accurate dosing for transplants and provides a critical tool for emerging gene-modified HSPC therapies where product characterization is paramount.