MYCT1 Controls Environmental Sensing to Maintain Self-Renewal in Human Hematopoietic Stem Cells

Abstract

Hematopoietic stem cells (HSC) sustain life-long blood production and have the ability to recapitulate the hematopoietic system upon transplantation. This makes them therapeutically invaluable for the treatment of hematopoietic malignancies and genetic disorders. However, the regulatory processes governing HSC functional competence and engraftment ability are poorly understood and challenging to recapitulate ex vivo, making robust expansion of human HSCs difficult to achieve. We have identified a novel HSC regulatory protein, MYCT1 (MYC target 1), that is highly enriched in self-renewing human HSC but lost during differentiation and culture. Knockdown (KD) experiments in human fetal liver and cord blood revealed a critical requirement for MYCT1 in human HSPC ex vivo expansion and engraftment across HSC ontogeny. Single cell RNAseq of human HSPCs upon MYCT1 KD linked MYCT1 loss in HLF+ HSCs to a profound dysregulation of cellular programs essential for HSC stemness, including mitochondrial activity, proteostasis and splicing. Because MYCT1 becomes drastically downregulated during HSC ex vivo culture, we investigated if restoring MYCT1 expression in culture could improve HSC function. Indeed, maintaining MYCT1 levels during culture via lentiviral expression resulted in improved “stemness” transcriptional signatures, greater expansion of the most undifferentiated EPCR+ITGA3+ human HSPCs, and enhanced engraftment ability upon transplantation. Notably, we also uncovered that the novel albumin free culture conditions reported by Sakurai et al. 2023 to robustly expand human HSCs can also maintain higher levels of MYCT1 expression. This reinforces our findings that the loss of MYCT1 expression is a major contributor to the functional incompetence of human HSCs cultured ex vivo. Nonetheless, the MYCT1 regulated processes and why they are critical for HSC competence are not known. Immunofluorescence and IP-mass spectrometry revealed that MYCT1 localizes in the endosomal membrane and interacts with vesicle trafficking machinery and receptor signaling components essential for HSC function. MYCT1 KD in human HSCs and endothelial cells (EC) led to hyperactive endocytosis and caused exaggerated signaling responses to cytokines in the culture microenvironment. We found that ex vivo culture gradually increased endocytosis specifically in CD34+ EPCR+ HSPCs in parallel with the culture-induced downregulation of MYCT1 in these cells, but not in the more differentiated progeny. On the other hand, restoring MYCT1 expression during HSC culture dampened the excessive endocytosis. Furthermore, maintaining low endocytosis during culture, either by the endocytosis inhibitor chlorpromazine or by sorting HSPCs with lower endocytic internalization of fluorescent dextran, results in the enrichment of immunophenotypic LT-HSCs in culture. Our data show that the moderation of environmental sensing through MYCT1-controlled endocytosis is essential for preserving human HSC self-renewal and engraftment ability. As MYCT1 expression is downregulated in cultured human HSPC, our findings imply that loss of the molecular machinery required for proper sensing of microenvironment signals has far-reaching effects into essential cellular functions required for stemness, and is a key contributor to the functional incompetence of cultured human HSC.