FBS Colloquia No.428Laboratory of Stem Cell Biology and Developmental Immunology

Identification of transcription factors necessary and sufficient for the formation of CAR cells that constitute the hematopoietic stem cell niche.

Fibroblasts are defined as non-hematopoietic, non-epithelial, non-endothelial, non-mesothelial, and non-parenchymal cells that produce extracellular matrix; however, the molecular basis underlying their diversity remains poorly understood. We previously demonstrated that Cxcl12-abundant reticular (CAR) cells in the bone marrow express high levels of CXCL12, an essential factor for hematopoietic stem cell (HSC) maintenance, and constitute a major component of the HSC niche. Based on their characteristic morphology and high expression of canonical fibroblast markers, including PDGFRα and vimentin, CAR cells are considered bone marrow-specific fibroblasts. More recently, tissue-specific fibroblasts that function as stem cell niches have also been identified in the intestine and lung, where they play essential roles in maintaining tissue homeostasis. In 2021, Turley and colleagues performed an integrated analysis of scRNA-seq datasets from fibroblasts across major tissues and proposed that fibroblasts can be divided into universal fibroblasts, which share transcriptional features across organs, and specialized fibroblasts, which acquire organ-specific functions. Their study further suggested that universal fibroblasts may give rise to specialized fibroblasts, implying a hierarchical organization among fibroblasts. Consistent with this concept, we demonstrated that universal fibroblasts isolated from the lung and colon can differentiate into CAR cells after transplantation into the bone marrow or muscle. Regardless of whether universal fibroblasts are physiological progenitors during development, these findings indicate that they possess the capacity to adopt diverse specialized fibroblast identities in response to tissue environments.
We therefore sought to identify transcription factors that are necessary and sufficient for CAR cell specification by using universal fibroblasts. We first overexpressed Foxc1 and Ebf3, transcription factors essential for the establishment and maintenance of the HSC niche function of CAR cells, in universal fibroblasts. However, the majority of cells failed to acquire a CAR cell phenotype, suggesting that additional transcription factors are required. We therefore performed scRNA-seq of embryonic day 17.5 femurs, in which CAR cells are undergoing specification, and identified 15 transcription factors that were preferentially expressed in developing CAR cells as candidate regulators.
In this colloquium, I will present our functional screening of these candidate transcription factors and reconstitution of HSC niche cells in vitro by these transcription factors.