Mouse BMSCs have been prospectively identified in the perivascular [53,103], while the populations usually lack expression of hematopoietic and endothelial markers but have positive expression of PDGFRα [11,38,39]. Later, Morikawa and Omatsu identified two distinctive subpopulations of PDGFRα⁺ BMSCs, PDGFRα⁺ Sca-1⁺ CD45⁻ Ter119⁻ BMSCs and PDGFRα⁺ Sca-1⁻ CD45⁻ Ter119⁻ BMSCs [11,38]. The former one resides primarily around arterioles but does not express the HSC niche factor Cxcl12, while the latter one resides primarily around sinusoids and expresses high levels of Cxcl12 to support hematopoiesis [11,38]. Similarly, another study found that Nestin-GFP⁺ BMSCs are heterogeneous, including both Nestin-GFP^high cells that localize mainly around arterioles and Nestin-GFP^low cells that localize mainly around sinusoids; both subpopulations can osteogenesis but Nestin-GFP^low cells secret more Cxcl12 [40,41]. Additionally, a previous study of Yue showed that LepR also marks SSPCs which localize in the perivascular region of the adult bone marrow [5] and promote osteogenesis [42], supporting the hematopoietic microenvironment by secreting high levels of Cxcl12 [5,43,44,45]. It has to be noticed that LepR⁺ BMSCs overlap with Nestin-GFP^low cells [40,41], Cxcl12-abundant reticular (CAR) cells [44], and Osterix⁺ BMSCs [39]. In contrast to LepR⁺ cells, Mx-1-Cre cells overlap with Nestin-GFP, PDGFRα, Sca1, and CD105 BMSCs and give rise to most of the osteoblasts formed in adult bone marrow, but Mx-1 also robustly labels hematopoietic cells, meaning that this marker could not be specific to selection of osteogenic progenitors population [39]. Moreover, Prx1 also is another important BMSC marker and overlaps with LepR⁺ stromal cells as well as LepR⁻ osteoblasts and chondrocytes within the bone marrow of limb bones, but not in the axial skeleton [46]. Most recently, Shu et al. found that Acan⁺ BMSCs and LepR⁺ BMSCs controlled bone formation before and after adolescence, respectively, and Acan⁺ BMSCs mediate bone lengthening, while LepR⁺ BMSCs regulate bone thickening [47]. In addition to the heterogeneity of BMSCs at different bone development stages, the heterogeneity of BMSCs from different bone regions has also received increasing attention. A recent publication proposes parathyroid hormone-related protein (PTHrP) as a label for chondrocytes of the resting zone in the growth plate of long bones which descend from a PTHrP⁺ SSCs [48]. Other findings also revealed that both Grem1⁺ cells and Gli1⁺ cells mainly reside in metaphyseal areas, Grem⁺ cells are non-adipogenic, while Gli1⁺ cells contribute to osteochondrogenesis and adipogenesis in vivo [49,50]. Later evidence suggested that Gli1 is also seen in many mature bone cell types (like, Osx, Col1) not uniquely marking an homogeneous BMSC population [50]. Recently, Sivaraj et al. characterized the heterogeneity of BMSCs during skeletal development. They identified that BMSCs from metaphysis and diaphysis have distinct properties, and the subpopulation of metaphyseal MSCs (mpMSCs) has multi-lineage differentiation potential to give rise to bone cells and LepR⁺ BMSCs, and transcription factors of platelet-derived growth factor B (PDGF-B) and Jun-B control BMSC osteogenesis [51]. Aside from growth plate and metaphyseal areas, many MSCs also reside along the periosteum, where they can quickly get “activated” upon injury and facilitate proper fracture healing [52].

Taking advantage of flow cytometry and scRNA-seq application, research on the heterogeneity of human BMSC subpopulations has also made good progress. In earlier studies, CD45 negative non-hematopoietic fibroblast colony-forming cells have been confirmed, and copious markers, including CD105, CD140a, CD73, CD90, STRO-1, CD271, and CD44 [7,8,9,10]. Another study reported that the CD146⁺ MSC subpopulation resides around sinusoidal blood vessels in the ossicles in the human bone marrow which can differentiate into osteogenic, chondrogenic, and adipogenic cells in culture and give rise to the bone upon transplantation in vivo, express HSC niche factors, and form bony ossicles that become invested with hematopoietic bone marrow [29,53]. Subsequent studies identified CD271⁺ PDGFRα^low and the stromal cell antigen 1(STRO-1⁺) turned out to most efficiently select for perivascular residing SSC-like cells that are also able to maintain human HSCs for an extended time in culture [54,55]. Groundbreaking experiments by Chan et al. have transplanted human single cells, termed bone cartilage stromal progenitor (BCSPs; CD45⁻ Ter119⁻ Tie2⁻ Thy1⁻ 6C3⁻ CD51⁺), to a renal capsule of mice, and confirmed the CD45⁻ Ter119⁻ Tie2⁻ Thy1⁻ 6C3⁻ CD51⁺ BCSPs were bone fide stem cells in vivo [22]. Currently, Chan et al. identified a highly purified human skeletal stem cell (hSSC) distinct from the reported CD146-positive SSCs; and found in fetal and all adult stages throughout different skeletal sites but specifically enriched in the hypertrophic zones of the growth plate labeled by PDPN, CD73, CD164 and lacking expression of CD235, CD45, CD146, Tie2, and CD31 [56], and the hSSC populations can give rise to osteoprogenitor cell types, never producing fat [15,57]. Moreover, Liu et al. have analyzed the BMSC heterogeneity of one healthy subject hip through sc-RNAseq, and they divided the BMSCs into three subpopulations [58]. Subpopulation A was characterized by the high expression of fibroblast growth factor receptor 2 (FGFR2) which was involved in osteogenesis; Subpopulation B expressed higher levels of fibroblast growth factor 5 (FGF5) which could increase osteogenic differentiation of MSCs; and Subpopulation C was characterized by high expression of plasminogen activator tissue type (PLAT) and vascular adhesion molecule 1(VCAM1) which promoted angiogenesis [58]. Similarly, Zhang et al. have proposed umbilical cord mesenchymal stem cells (UC-MSCs) having two subpopulations via sc-RNAseq [59]. Group 1 MSCs are enriched in the expression of genes in immune response/regulatory activities (e.g., TNFα, IL17, TLR, TGFβ, infection, NOD, NF-κB, and PGE pathways), muscle cell proliferation and differentiation, stemness, and oxidative stress while group 2 MSCs are enriched with gene expression in extracellular matrix production, bone, and cartilage growth as well glucose metabolism [59].

In sum, human BMSC subpopulations possess more complex cell surface markers, exhibit specific bone regions distributions and different differentiation potential, and the subpopulations from the hip and umbilical cord show distinctive functional heterogeneity (Table 1). In detail, human BMSCs express copious markers, including CD105, CD140a, CD73, CD90, STRO-1, CD271, and CD44 et al., which exhibited variable CFU-F ability and multi-lineage differentiation potential. CD146⁺, CD271⁺ PDGFRα^low, and STRO-1⁺ BMSC subpopulations contribute to HSC niche stability. CD45⁻ Ter119⁻ Tie2⁻ Thy1⁻ 6C3⁻ CD51⁺ labels BCSPs; while PDPN⁺ CD73⁺ CD164⁺ CD235⁻ CD45⁻ CD146⁻ Tie2⁻ CD31⁻ labels BMSCs, residing in hypertrophic zones of the growth plate, and these subpopulations just give rise to osteoprogenitor cell types, never producing fat. In addition, BMSCs of the healthy subject can be divided into FGFR2⁺; FGF5⁺ BMSCs and PLAT⁺ VCAM1⁺ BMSCs promote osteogenesis and angiogenesis, respectively. Similarly, UC-MSCs also can be divided into immune response/regulatory activities related to MSC group 1 and bone and cartilage growth-related MSC group 2. Importantly, human BMSC subpopulations showed distinctive markers with mouse BMSCs.

Both the number and function of BMSCs decline dramatically with aging. Thus, it is very important to identify exact BMSC subpopulations that dismiss or increase during aging. Recent studies have been devoted to identifying some elderly BMSC subpopulations. Liu et al. have reported that a Sca1⁺ BMSC subpopulation from aged mice exhibited lower paracrine support for retinas than a Sca1⁺ BMSC subpopulation from young mice [60], and injecting young Sca-1⁺ BMSCs into 18-month-old mice through the tail vein can increase brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), FGF2, and insulin-like growth factor 1 (IGF-1) expression, downregulation of the apoptotic protein Bax with upregulation of the antiapoptotic protein Bcl2 to attenuate aging-related retinal degeneration ultimately [60]. Other studies have indicated impaired osteogenic potential of Prx1⁺ SSCs in aging mice because the loss of the peroxlsome proliferator-activated receptor-g coactivator-1 a (PGC-1α), an expression in aging Prx1⁺ SSCs, and overexpression of PGC-1α in SSCs reversed the unbalance of SSC osteo-adipogenic differentiation during aging [61]. Wang et al. have confirmed that alpha-ketoglutarate (αKG) can rejuvenate the osteogenic capacity of LepR⁺ BMSCs and ameliorate age-related osteoporosis by decreasing the accumulations of H3K9me3 and H3K27me3, and subsequently upregulates BMP signaling and Nanog expression [62]. Recently, a study by Yue revealed that LepR⁺ BMSCs were decreased with aging and enhanced adipogenesis in bone marrow, and downregulated HSC-supportive Kitl and Cxcl12 expression derived from LepR⁺ Notch3⁺ BMSCs [63]. Zhong et al. have found that the great expansion of LepR⁺ marrow adipogenic lineage precursors (MALPs) in 16-month-old mice might explain why there is more adipose tissue accumulation with aging [19].

In addition to aged mice, the study of Zhu et al. recently investigated the heterogeneity of BMSCs (CD29⁺ CD44⁺ CD90⁺ CD105⁺ CD34⁻ CD45⁻ HLA-DR⁻) from an 85-year-old human by sc-RNAseq [64]. They divided the BMSCs populations into three clusters according to the expression of functional genes [64]. Cluster 1 enriched the expression of cell self-renewal including cell division cycle-associated 5 (CDCA5) [65] and V-Myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2) [66], the cell metaphase–anaphase transition-related regulator family with sequence similarity 64 (FAM64A) [67], the integrity of the human centromere DNA repeats related to centromere protein A (CENP-A) [68], cell proliferation-related progestin and adipoQ receptor family member 4 (PAQR4) [69], DNA replication and repair related histone chaperone anti-silencing function 1B (Asf1b) [70], and chromatin assembly factor-1 (CAF-1) [71] and high mobility group protein 2 antibody (HMGB2) [72]. Cluster 2 is high expression multidirectional differentiation of BMSCs, including chondrogenesis-related transglutaminase 2 (TGM2) [73] and COL11A1 [74], osteogenesis-related nuclear factor of activated T-cells (NEAT1) [75] and Type V collagen [76]. Cluster 3 enriched the expression of secretory factors to participate in immune regulation and damage repair including microbial killing and innate immunity-related Cyba [77], regulation of immunity and inflammation-related tissue inhibitor of metalloproteinase-1 (TIMP-1) [78] and annexin A1 (ANXA1) [79], cell migration and wound repair-related lumican (LUM) [80], and dermatopontin (DPT) [81], regulating cytokine secretion-related endoplasmic reticulum resident protein 44 (ERp44) [82], and protein import into endoplasmic reticulum-related heat shock protein family A, member 5 (HSPA5) [83].

Taken together, specific BMSC subpopulations exhibit changed numbers and impaired osteogenesis, and enhanced adipogenesis during aging (Table 1). For example, Sca1⁺, Prx1⁺, LepR⁺, LepR⁺ Notch3⁺ BMSCs of aging mice were all declined and exhibited impaired paracrine support for retinas and HSC niche, while LepR⁺ MALPs were increased in aging mice and contributed to adipogenesis. Moreover, elderly human BMSC populations can be divided three populations, including self-renewal, multidirectional differentiation, and immune regulation and damage repair.

Apart from the different development stage, the heterogeneous BMSCs are also important for bone to react to different environmental stresses.

2.1. Loading

2.2. Microgravity

2.3. Hypoxia

2.4. Irradiation

2.5. PTH