Endothelial Expansion Contributes to Enhanced Blood Cell Production from Tropomyosin 1 Deficient Induced Pluripotent Stem Cells

Background: Donor-derived blood products are critical to our healthcare system, but do not fully meet the needs of all patients. For example, preterm infants suffer increased morbidity and mortality from allogeneic platelet transfusions. Ex vivo production of more appropriate ‘embryonic’ or ‘fetal type’ blood cells could address transfusion-related issues, but efforts to derive blood cells from induced pluripotent stem cells (iPSCs) in vitro are inefficient.

We previously used machine learning-based analysis of human genetic studies, and genome-edited iPSC models, to determine that Tropomyosin 1 (TPM1) inhibits in vitro hematopoiesis. TPM1 knockout (TPM1KO) iPSCs produced 2-fold more hematopoietic progenitor cells (HPCs) than controls, thereby increasing production of mature blood cells that were functionally normal (Thom et al, BMC Biol 2020). Here, we aimed to i) define related cellular and molecular mechanisms and ii) reveal novel targets that may be pharmacologically manipulated to enhance blood cell production without need for genomic manipulation.

Methods: We interrogated wild type and isogenic TPM1KO iPSCs during primitive (embryonic) hematopoietic differentiation using cell and molecular biology techniques. In this in vitro differentiation system, cells adopt mesoderm, endothelial, and hematopoietic identities through sequential epithelial-to-mesenchymal transitions (EMTs). Each cell type has distinct surface markers that can be used for identification and isolation.

Results: We hypothesized that TPM1KO may enhance cell proliferation and/or survival at one or more developmental transitions during in vitro differentiation. By cell counting and flow cytometric analyses, TPM1KO cells were not hyperproliferative as iPSCs or at any stage of differentiation. Instead, more TPM1KO cells survived EMTs at the mesoderm-to-endothelial and/or endothelial-to-hematopoietic transition points. This resulted in an increased population of endothelial cells; ‘hemogenic’ endothelial cells within this population are responsible for increased HPC yield.

To define molecular mechanisms by which TPM1 regulates EMTs during in vitro primitive hematopoiesis, we performed RNA sequencing analysis on sorted iPSCs, endothelial cells, and HPCs from TPM1KO and control cultures. TPM1KO endothelial cells and HPCs had altered expression of genes and pathways known to promote EMT, including altered cell adhesion, integrin expression, and integrin-mediated signaling genes (p < 0.05). For example, TPM1KO cells showed increased expression of N-cadherin and RAP1-activating genes. Increased N-cadherin and activated RAP1 facilitate cellular epithelial-to-mesenchymal transitions in other biological contexts.

Conclusion: Our findings show that TPM1KO enhances in vitro endothelial cell production through increased EMT survival. In other words, TPM1KO cells undergo a more efficient differentiation, which enhances total HPC production. TPM1-mediated EMT regulation represents a novel mechanism that may be exploited to augment in vitro blood cell yield, through RAP1 or other pharmacologically modifiable pathways. Application of these findings may help to boost in vitro blood product generation production toward clinically relevant scales.

This work was supported by a 2019 American Academy of Pediatrics Marshall Klaus Perinatal Research Award.