Manipulation of Tropomysin 1 in iPSCs to Enhance in Vitro Blood Cell Production

Donor-derived blood transfusions are critical to our healthcare system, but do not fully meet the needs of patients with multiple alloantibodies, rare blood types, or HLA-sensitization. These needs have fueled the concept of ex vivo blood cell production, which might address issues related to demographic aging, infectious outbreaks transmitted by transfusions, and rare blood types. Blood cells produced in vitro could be used for transfusions, and could also be used as blood bank testing reagents (Coleman et al, Transfusion 2019). One major challenge in deploying this system is efficiently scaling up cell production.

We used machine learning and genome-edited induced pluripotent stem cell (iPSC) models to determine that Tropomyosin 1 (TPM1) normally 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). During human hematopoiesis, HPCs arise from specialized vascular 'hemogenic endothelial' cells (HE) with distinct surface markers that can be used for identification and isolation. To define molecular mechanisms by which TPM1 regulates in vitro primitive hematopoiesis, we performed RNA sequencing analysis on sorted KDR⁺CD31⁺ endothelial cells and CD43⁺ HPCs from TPM1KO and control cultures. TPM1KO endothelial cells and HPCs had altered expression of genes and pathways known to regulate HE biology, including cell adhesion, integrin expression, and integrin-mediated signaling (p<0.05). 'Anoikis' is an apoptosis-like programmed cell death that occurs after extracellular matrix detachment. This process may limit nascent non-adherent HPC production in vitro, but has not been previously studied. TPM1KO cells showed increased expression of N-cadherin and RAP1-activating genes; increased N-cadherin and activated RAP1 limit anoikis in other biological contexts. In sum, these results suggested that TPM1KO cultures increased HE production and/or survival. To analyze HE production at the single cell level, we sorted KDR⁺CD31⁺CD43^- endothelial cells and plated them in limiting dilution. We cultured sorted cells in hematopoietic cytokines for 7 days, and analyzed the number of wells in which CD43⁺ HPCs arose from sorted TPM1KO and control cells. Using limiting dilution analysis (Hu & Smyth,J. Immunol. Methods 2009), we found that TPM1KO cultures produced 2-fold more HE than controls.

These results show that TPM1KO enhances in vitro hematopoiesis by increasing HE and subsequent HPC production, perhaps by limiting anoikis in nascent HPCs. TPM1-mediated regulation at the HE stage represents a novel mechanism that may be genetically or pharmacologically exploited to augment in vitro hematopoiesis. These findings will help boost in vitro HPC and blood cell production to clinically relevant scales, supporting efforts to produce blood cells for direct transfusion and/or to be used as clinical screening reagents.