337 - Endothelial expansion contributes to enhanced blood cell production from Tropomyosin 1 deficient induced pluripotent stem cells

Poster Number: 337
Publication Number: 337.122

Madison B. Wilken, Childrens Hospital of Philadelphia, Philadelphia, PA, United States; Benjamin F. Voight, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States; Stella T. Chou, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States; Deborah L. French, Children's Hospital of Philadelphia, Philadelphia, PA, United States; Christopher S. Thom, Children's Hospital of Philadelphia, Philadelphia, PA, United States

Background: Donor-derived blood products do not currently meet the needs of all patients, including preterm infants who suffer increased morbidity and mortality from allogeneic platelet transfusions. In vitro-derived ‘embryonic’ or ‘fetal’ type blood cells from induced pluripotent stem cells (iPSCs) could address these issues, but current production strategies are inefficient.
We previously found that Tropomyosin 1 (TPM1) inhibits in vitro hematopoiesis (Thom et al, BMC Biol 2020). Genome-edited TPM1 knockout (TPM1KO) iPSCs produced 2-fold more hematopoietic progenitor cells (HPCs) than controls, thereby increasing functionally normal mature blood cell yields.

Objective: Here, we aimed to i) define related mechanisms and ii) reveal pharmacologically manipulatable targets to enhance blood cell production without need for genomic modification.

Design/Methods: We used cell and molecular biology techniques to interrogate wild type and isogenic TPM1KO iPSCs during primitive (embryonic) hematopoietic differentiation. In this in vitro differentiation system, iPSCs sequentially adopt mesoderm, endothelial, and hematopoietic identities through epithelial-to-mesenchymal transitions (EMTs). Distinct surface markers allow identification and isolation of each cell type.

Results: We used cell counting and flow cytometry to determine if TPM1KO enhanced cell proliferation and/or survival at each developmental stage. TPM1KO cells were not hyperproliferative as iPSCs or at any differentiation stage. Instead, TPM1KO enhanced survival during mesoderm-to-endothelial and endothelial-to-hematopoietic transitions. This quantitatively increased total endothelial cell production, and ‘hemogenic’ endothelial cells within this expanded population were responsible for increased HPC yields.
To define molecular mechanisms by which TPM1 regulates EMTs during in vitro hematopoiesis, we analyzed RNA expression in sorted iPSCs, endothelial cells, and HPCs from TPM1KO and control cultures. TPM1KO altered expression of genes and pathways known to promote EMT,
including cell adhesion, integrin, and integrin-signaling gene expression (p < 0.05). For example, TPM1KO endothelial cells had increased N-cadherin and RAP1-activating gene expression, which promote EMT in other cellular and biological contexts.Conclusion(s): TPM1KO cells undergo more efficient differentiation, increasing in vitro endothelial cell and ultimately blood cell production. Exploiting TPM1-mediated EMT regulation, through RAP1 or other pharmacologically modifiable pathways, represents a novel strategy to boost in vitro blood cell production toward clinically relevant scales.