Radial extracorporeal shockwave (r-ESW) and bone marrow stromal cells (BMSCs) have been reported to alleviate senile osteoporosis (SOP), but its regulatory mechanism remains unclear. In this study, we firstly isolated human BMSCs from bone marrow samples and treated with varying r-ESW doses. And we found that r-ESW could enhance the proliferation of SOP-BMSCs in a dose-dependent manner by EdU assay. Subsequently, the impact of r-ESW on the proliferation, apoptosis and multipotency of BMSCs was assessed. And the outcomes of flow cytometry, Alizarin red S (ARS), and tube formation test demonstrated that the optimal shockwave obviously boosted SOP-BMSCs osteogenesis and angiogenesis but exhibited no significant impact on cell apoptosis. Additionally, the signaling of Piezo1 and CaMKII/CREB was examined by Western blotting, qPCR and immunofluorescence. And the results showed that r-ESW promoted the expression of Piezo1, increased intracellular Ca2+ and activated the CaMKII/CREB signaling pathway. Then, the application of Piezo1 siRNA hindered the r-ESW-induced enhancement ability of osteogenesis coupling with angiogenesis of SOP-BMSCs. The use of the CaMKII/CREB signaling pathway inhibitor KN93 suppressed the Piezo1-induced increase in osteogenesis and angiogenesis in SOP-BMSCs. Finally, we also found that r-ESW might alleviate SOP in the senescence-accelerated mouse prone 6 (SAMP6) model by activating Piezo1. In conclusion, our research offers experimental evidence and an elucidated underlying molecular mechanism to support the use of r-ESW as a credible rehabilitative treatment for senile osteoporosis.

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME\'s intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.

With the aging of the global demographic, the prevention and treatment of osteoporosis are becoming crucial issues. The gradual loss of self-renewal and osteogenic differentiation capabilities in bone marrow stromal cells (BMSCs) is one of the key factors contributing to osteoporosis. To explore the regulatory mechanisms of BMSCs differentiation, we collected bone marrow cells of femoral heads from patients undergoing total hip arthroplasty for single-cell RNA sequencing analysis. Single-cell RNA sequencing revealed significantly reduced CRIP1 (Cysteine-Rich Intestinal Protein 1) expression and osteogenic capacity in the BMSCs of osteoporosis patients compared to non-osteoporosis group. CRIP1 is a gene that encodes a member of the LIM/double zinc finger protein family, which is involved in the regulation of various cellular processes including cell growth, development, and differentiation. CRIP1 knockdown resulted in decreased alkaline phosphatase activity, mineralization and expression of osteogenic markers, indicating impaired osteogenic differentiation. Conversely, CRIP1 overexpression, both in vitro and in vivo, enhanced osteogenic differentiation and rescued bone mass reduction in ovariectomy-induced osteoporosis mice model. The study further established CRIP1\'s modulation of osteogenesis through the Wnt signaling pathway, suggesting that targeting CRIP1 could offer a novel approach for osteoporosis treatment by promoting bone formation and preventing bone loss.

Bone marrow stromal cells (BMSCs) can promote the growth of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). Histone deacetylases (HDACs) play essential roles in the proliferation and apoptosis resistance of Ph + ALL cells. In our previous study, inhibiting histone deacetylase 1 (HDAC1) decreases the proliferation of Ph + ALL cells. However, little is known regarding how HDAC1 in BMSCs of Ph + ALL patients affects the imatinib (IM) resistance. Therefore, the present work examined the roles of HDAC1 in BMSCs. Overexpression of HDAC1 was found in BMSCs of Ph + ALL patients with IM resistance. In addition, the Ph + ALL cell line SUP-B15 was co-cultured with BMSCs after lentivirus transfection for regulating HDAC1 expression. Knockdown of HDAC1 within BMSCs elevated the IM-mediated SUP-B15 cell apoptosis, while increasing HDAC1 expression had an opposite effect. IL-6 in BMSCs, which is an important factor for the microenvironment-associated chemoresistance, showed evident up-regulation in HDAC1-upregulated BMSCs and down-regulation in HDAC1-downregulated BMSCs. While recombinant IL-6 (rIL-6) can reversed the sensitivity of SUP-B15 cells to IM induced by downregulating HDAC1 expression in BMSCs. HDAC1 showed positive regulation on IL-6 transcription and secretion. Moreover, IL-6 secretion induced by HDAC1 in BMSCs might enhance IM resistance in Ph + ALL cells. With regard to the underlying molecular mechanism, NF-κB, an important signal responsible for IL-6 transcription in BMSCs, mediated the HDAC1-regulated IL-6 expression. Collectively, this study facilitated to develop HDAC1 inhibitors based not only the corresponding direct anti-Ph + ALL activity but also the regulation of bone marrow microenvironment.

The differentiation of bone marrow stromal cells (BMSCs) into Schwann-like cells (SCLCs) has the potential to promote the structural and functional restoration of injured axons. However, the optimal induction protocol and its underlying mechanisms remain unclear. This study aimed to compare the effectiveness of different induction protocols in promoting the differentiation of rat BMSCs into SCLCs and to explore their potential mechanisms. BMSCs were induced using two distinct methods: a composite factor induction approach (Protocol-1) and a conditioned culture medium induction approach (Protocol-2). The expression of Schwann cells (SCs) marker proteins and neurotrophic factors (NTFs) in the differentiated cells was assessed. Cell proliferation and apoptosis were also measured. During induction, changes in miR-21 and Sprouty RTK signaling antagonist 2 (SPRY2) mRNA were analyzed. Following the transfection of BMSCs with miR-21 agomir or miR-21 antagomir, induction was carried out using both protocols, and the expression of SPRY2, ERK1/2, and SCs marker proteins was examined. The results revealed that NTFs expression was higher in Protocol-1, whereas SCs marker proteins expression did not significantly differ between the two groups. Compared to Protocol-1, Protocol-2 exhibited enhanced cell proliferation and fewer apoptotic and necrotic cells. Both protocols showed a negative correlation between miR-21 and SPRY2 expression throughout the induction stages. After induction, the miR-21 agomir group exhibited reduced SPRY2 expression, increased ERK1/2 expression, and significantly elevated expression of SCs marker proteins. This study demonstrates that Protocol-1 yields higher NTFs expression, whereas Protocol-2 results in stronger SCLCs proliferation. Upregulating miR-21 suppresses SPRY2 expression, activates the ERK1/2 signaling pathway, and promotes BMSC differentiation into SCLCs.