Abstract Stromal progenitor cells of bone marrow origin are non-hematopoietic cells that give rise to osteoblasts and adipocytes in the postnatal organism. Marrow stromal cells (MSCs) (also known as skeletal stem cells)are currently being employed in a large number of clinical trials for regenerative purposes post in vitro expansion. However, the clinical outcome has been variable, which might in part be due to the heterogeneity of the cells and the lack of a defined cell product with a molecular signature that favors tissue regeneration. In this study, we determined the cellular heterogeneity of primary stromal cultures and examined how inter-donor variation in subpopulation composition contributes to the differentiation potential of primary cultures. We profiled 136,014 stromal progenitors from 26 donors and identified 5 subpopulations that were linked to distinct bone-related pathways and genetic traits of bone mineral density and morphology. Abundancy of one cluster characterized by high expression of ITGA11 and genes related to matrix function, collagen organization, and elevated expression upon lineage commitment was positively correlated with osteoblastic differentiation capacity in vitro. In addition, ITGA11 protein expression in progenitor cells was a predictive marker for matrix mineralization in vitro and ectopic bone formation in vivo. Sorting stromal progenitors into ITGA11high and ITGA11low cells established cultures with high and low osteoblastic differentiation potential, respectively. Our findings corroborate the presence of extensive cellular heterogeneity among cultured human stromal cells, which strongly differs from donor to donor, and that ITGA11 can be employed as a marker for isolating cells with high bone-forming potential, a feature likely to benefit clinical trials of bone regeneration.

This study aimed to investigate the therapeutic effect of human nasal turbinate-derived stem cells (hNTSCs) on mice with rheumatoid arthritis (RA) and identify hNTSC gene signatures with therapeutic effects on RA. hNTSCs were obtained from 20 healthy controls (HCs) who had undergone nasal turbinate surgery. Collagen-induced arthritis (CIA) mice were used to investigate the therapeutic effects of hNTSCs. The engraftment and migration abilities of hNTSCs were evaluated. CD4+CD25- T cells were co-cultured with hNTSCs, and effector T cell proliferation was evaluated by flow cytometry. Osteoclast differentiation was evaluated using mouse bone marrow monocytes which were cultured with M-CSF and RANKL, then TRAP staining was performed to measure effect of hNTSCs on osteoclastogenesis. Microarray assays were performed to identify gene expression differences between hNTSCs with CIA mice therapeutic or not and were validated by RT-qPCR. hNTSCs differentiated well into osteoblasts and adipocytes and expressed high levels of CXCL1 and osteoprotegerin. Single-cell RNA sequencing showed that hNTSCs clustered into 11 cell types, and cell surface markers were compatible with mesenchymal stem cells. hNTSC-treated CIA mice showed reductions in arthritis severity scores and incidence of arthritis. In engraft measurements, hNTSCs survived for 8 to 12 weeks in mice paws. Chemokine receptors expression increased in hNTSCs by IL-1β or TNF-α stimulation. CD4+CD25- T cell proliferation was reduced by hNTSCs and reversed by adding 1-MT (indoleamine 2,3-dioxygenase inhibitor), indicating that indoleamine 2,3-dioxygenase mediated T cell suppression. Osteoclastogenesis was suppressed by hNTSCs, and this was attenuated by anti-OPG Ab. hNTSCs therapeutic in CIA mice showed specific gene signatures with up-regulated genes (KRTAP1-5, HAS2, and CXCL1) and down-regulated genes (GSTT2B and C4B) compared to hNTSCs without CIA therapeutic effects. hNTSCs exhibited therapeutic potential in RA. Therapeutic effects were mediated by effector helper T cell suppression and the inhibition of osteoclastogenesis. In addition, hNTSCs with greater therapeutic effects on RA showed significant differences in their gene signatures.
© 2025. The Author(s).

Bone marrow adipose tissue (BMAT) has garnered significant attention due to its critical roles in leukemia pathogenesis, cancer metastasis, and bone marrow failure. BMAT is a metabolically active, distinct tissue that differs from other fat depots. Marrow adipocytes, closely interacting with hematopoietic stem/progenitor cells and osteoblasts, play a pivotal role in regulating their functions. However, standardized methods for isolating and defining human BMAT (hBMAT) remain unclear. In animal models, BMAT is commonly isolated directly from the bone marrow through flushing, enzymatic digestion, or mechanical disruption. In humans, BMAT isolation often involves the adipogenic induction of bone marrow mesenchymal stem/stromal cells (BM-MSCs) derived from bone marrow aspirates. However, this approach reflects cellular responses to chemical stimuli and may not accurately represent in vivo differentiation potential. Similarly, BMAT obtained from hip or knee replacement surgeries might not reflect basal physiological conditions due to inflammatory influences. Here, we describe a direct method for culturing BMAT from the fatty layer of bone marrow aspirates obtained from healthy transplant donors. This protocol employs centrifugation and washing steps using basic laboratory equipment, offering simple and non-enzymatic approach. For validation, isolated cells are characterized according to the International Society for Cell & Gene Therapy (ISCT) criteria. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human BMAT-MSCs from the fatty layer of the bone marrow Basic Protocol 2: Culture expansion, trypsinization, and cryopreservation of BMAT-MSCs Support Protocol 1: Immunophenoypic characterization of human BMAT-MSCs by flow cytometry Support Protocol 2: In vitro characterization of multilineage differentiation potential of human BMAT-MSCs Support Protocol 3: Further characterization of gene expression in human BMAT-MSCs using qRT-PCR.
© 2025 Wiley Periodicals LLC.

PIK3CA-related overgrowth spectrum (PROS) encompasses several rare conditions that lead to overgrowth of various body parts resulting from activating variants in PIK3CA. The absence of ideal cell models significantly impedes progress in PROS research. In this study, we focused on facial infiltrating lipomatosis (FIL) (A disorder within PROS) and aimed to establish and characterize an immortalized PROS cell line. Primary adipose-derived stem cells of FIL were immortal-ized through the transfection of simian virus 40 large T antigen (SV40LT). No significant mor-phological differences were observed in immortalized FIL-ADSCs (Im FIL-ADSCs). Im FIL-ADSCs expressed original mesenchymal surface markers, confirmed by flow cytometry. It harbored PIK3CA mutation and an increased level of PI3K/AKT activation, revealed by sanger sequencing and Western blot respectively. Karyotype analysis revealed a stable chromosome in Im FIL-ADSCs. Higher adipogenic potential and lower osteogenic differentiation properties were de-tected in Im FIL-ADSCs. The proliferative potential of Im FIL-ADSCs increased, whereas malig-nant transformation was not observed in the tumorigenesis assay. Moreover, RNA sequencing further elucidated the role of the transcription factor E2F1 in Im FIL-ADSCs. Drug screening unveiled that STAT3, HSP, EGFR, and NF-kB might be potential therapeutic targets for FIL. This study provided a valuable cellular resource for exploring the underlying pathogenic mechanisms and developing new targeted therapeutic options for PROS.
© 2024 Published by Elsevier B.V.

The ovary is crucial for female reproduction and health, as it generates oocytes and secretes sex hormones. Transplantation of mesenchymal stem cells (MSCs) has been shown to alleviate pathological ovarian aging. However, it is unclear whether MSCs could benefit the naturally aging ovary. In this study, we first examined the dynamics of ovarian reserve of Chinese women during perimenopause. Using a naturally aging cynomolgus monkey (Macaca fascicularis) model, we found that transplanting human embryonic stem cells-derived MSC-like cells, which we called M cells, into the aging ovaries significantly decreased ovarian fibrosis and DNA damage, enhanced secretion of sex hormones and improved fertility. Encouragingly, a healthy baby monkey was born after M-cell transplantation. Moreover, single-cell RNA sequencing analysis and in vitro functional validation suggested that apoptosis, oxidative damage, inflammation, and fibrosis were mitigated in granulosa cells and stromal cells following M-cell transplantation. Altogether, these findings demonstrate the beneficial effects of M-cell transplantation on aging ovaries and expand our understanding of the molecular mechanisms underlying ovarian aging and stem cell-based alleviation of this process.
© 2024. The Author(s).