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Mesenchymal stromal cell (MSC) differentiation is critical for the development, maintenance, and repair of bone tissue. MSCs also play a key role in regulating self-renewal and differentiation of normal hematopoietic and leukemic stem cells. Our prior work has identified a key role of taurine produced by bone marrow osteolineage cells in supporting the growth of taurine transporter (TauT or Slc6a6) expressing leukemia cells. Here, we analyze multiple murine non-hematopoietic bone marrow single-cell RNA-sequencing datasets and discover that TauT expression is enriched in MSCs in vivo. Although taurine supplements have been shown to mitigate bone defects in aged mice, its role in regulating MSC populations that give rise to bone cells is poorly understood. Using TauT genetic loss-of-function murine models, we find that TauT loss impacts murine MSC populations in vivo and impairs MSC osteogenic differentiation in vitro. This is associated with decreased bone mineral density and bone strength in young and aged TauT knockout mice. Importantly, shRNA-based knockdown of TAUT expression in primary human donor MSCs reduces osteogenic differentiation. TauT null MSCs are unable to support self-renewal and expansion of co-cultured hematopoietic stem and progenitor populations, indicating broad functional defects. Mechanistically, TauT loss results in downregulation of inositol metabolism, increased oxidative stress, and reduced Wnt/β-catenin signaling, which induce MSC senescence. Collectively, our data identifies taurine as a key regulator of MSC maintenance and osteogenic fate determination.

Simple SummaryCancer-associated fibroblasts (CAFs) play a crucial role in the tumor stroma. Our RNA sequencing analysis with 22 CAF and 11 normal fibroblast (NF) samples of non-small-cell carcinoma (NSCLC) revealed specific CAF markers. COL11A1, GREM1, CD36, and GAS6 are highly expressed in CAFs. Both GREM1 and GAS6 showed a strong expression in CAFs from lymph nodes and CAFs from lung specimens relative to NFs. TNC and CXCL2 are prominent in NFs. Differential expression patterns were observed in lymph node and lung specimens. In the co-culture model of CAFs and THP-1 cells, the knockdown of GREM1 or GAS6 in CAFs significantly decreased the M2 marker expression in macrophages. In NSCLC, GREM1 and GAS6 represent potential diagnostic targets for CAFs derived from both primary tumors and metastatic sites. AbstractBackground/Objectives: Cancer-associated fibroblasts (CAFs) play a pivotal role in the tumor microenvironment. We conducted an analysis using RNA sequencing to identify specific markers for CAFs compared to normal fibroblasts (NFs) in non-small-cell carcinoma (NSCLC). Methods: CAFs and NFs were isolated and cultured from tumor tissues (primary tumor or metastatic lymph nodes) and matched non-tumor tissues, respectively. Bulk RNA sequencing was conducted on isolated CAFs and normal fibroblast NFs. Differential expressions, gene set enrichment, and CAF subpopulation prediction analyses were performed. Results: During the study period, 27 CAFs and 12 NFs were isolated and cultured from tumor and non-tumor tissues in patients with treatment-naïve NSCLC. Among them, 22 CAFs and 11 NFs were included in the RNA sequencing analysis. The 22 CAF samples consisted of 12 adenocarcinomas and 10 squamous cell carcinomas (SqCC), with 16 samples from the lungs and 6 samples from the lymph nodes. Notably, COL11A1, GREM1, CD36, and GAS6 showed a higher expression in CAFs than in NFs, whereas TNC and CXCL2 were more abundantly expressed in NFs. CD36 levels were elevated in CAFs from lymph nodes (LN-CAFs) compared with those from lung specimens (Lung-CAFs) and NFs. COL11A1 levels in Lung-CAFs surpassed those in LN-CAFs and NFs. Both GREM1 and GAS6 showed a strong expression in Lung-CAFs and LN-CAFs relative to NFs. CAFs exhibited features of the myofibroblast CAF subpopulation, whereas NFs displayed traits of the antigen-presenting CAF subtype. In the co-culture model of CAFs and THP-1 cells, the knockdown of GREM1 or GAS6 in CAFs significantly decreased the M2 marker expression in macrophages. Conclusions: In NSCLC, GREM1 and GAS6 can be valuable diagnostic targets for CAFs from primary tumors and metastatic sites; they warrant further study.

AbstractStromal cells are critical regulators of hematopoietic stem/progenitor cells and skeletal homeostasis. Although precise systems for functional analysis are critical to investigate mechanistically bone and bone marrow (BM)-derived stromal cells, the establishment of reproducible, highly enriched ex vivo methods for stromal cell isolation, culture and evaluation have been challenging, leading to inconsistent data on stromal cell function. In this work, we carefully tested ex vivo culture of murine stromal cells from BM and bone and discovered abundant and persistent contamination of monocytes and macrophages. We succeeded in establishing highly enriched ex vivo culture system for stromal cells by eliminating persistent monocytes and macrophages using selection against the immunological markers F4/80, Ly6C, and CD45. Transcriptional and functional assays of enriched stromal cell culture revealed differential characteristics of stromal cells from different origins, a dormant signature for bone-derived cells and a highly proliferative progenitor-like signature for BM-derived cells. Monocyte and macrophage contamination reduced signatures of immature stromal cells such as expression levels of SOX9 and CD140a as well as the cells’ ability to support hematopoietic stem and progenitor cells based on our growth factor-free co-culture system of hematopoietic cells and stromal cells followed by in vivo functional assays. The inhibitory effects of macrophages on stromal cells may be explained by their potent production of inflammatory cytokines such as CXCL2, CCL3, and complement factor (C1q) confirmed by protein immunoassay of culture supernatant, as well as the differential contribution of pre-osteoblasts to the stromal cell population. This study highlights the functional diversity of stromal cells depending on the microenvironment of origin while addressing a critical limitation of murine ex vivo systems. Our robust culture system enables the study of isolated stromal cells function as well as the impact of stromal cells-macrophage crosstalk.