Distinct skeletal stem cell types orchestrate long bone skeletogenesis.
Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research.
View details for DOI 10.7554/eLife.66063
View details for PubMedID 34280086
Aged skeletal stem cells generate an inflammatory degenerative niche.
Loss of skeletal integrity during ageing and disease is associated with an imbalance in the opposing actions of osteoblasts and osteoclasts1. Here we show that intrinsic ageing of skeletal stem cells (SSCs)2 in mice alters signalling in the bone marrow niche and skews the differentiation of bone and blood lineages, leading to fragile bones that regenerate poorly. Functionally, aged SSCs have a decreased bone- and cartilage-forming potential but produce more stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell RNA-sequencing studies link the functional loss to a diminished transcriptomic diversity of SSCs in aged mice, which thereby contributes to the transformation of the bone marrow niche. Exposure to a youthful circulation through heterochronic parabiosis or systemic reconstitution with young haematopoietic stem cells did not reverse the diminished osteochondrogenic activity of aged SSCs, or improve bone mass or skeletal healing parameters in aged mice. Conversely, the aged SSC lineage promoted osteoclastic activity and myeloid skewing by haematopoietic stem and progenitor cells, suggesting that the ageing of SSCs is a driver of haematopoietic ageing. Deficient bone regeneration in aged mice could only be returned to youthful levels by applying a combinatorial treatment of BMP2 and a CSF1 antagonist locally to fractures, which reactivated aged SSCs and simultaneously ablated the inflammatory, pro-osteoclastic milieu. Our findings provide mechanistic insights into the complex, multifactorial mechanisms that underlie skeletal ageing and offer prospects for rejuvenating the aged skeletal system.
View details for DOI 10.1038/s41586-021-03795-7
View details for PubMedID 34381212
Articular cartilage regeneration by activated skeletal stem cells.
Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage1. The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation2-4. Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis5-7. We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.
View details for DOI 10.1038/s41591-020-1013-2
View details for PubMedID 32807933
Geriatric fragility fractures are associated with a human skeletal stem cell defect.
Fragility fractures have a limited capacity to regenerate, and impaired fracture healing is a leading cause of morbidity in the elderly. The recent identification of a highly purified bona fide human skeletal stem cell (hSSC) and its committed downstream progenitor cell populations provides an opportunity for understanding the mechanism of age-related compromised fracture healing from the stem cell perspective. In this study, we tested whether hSSCs isolated from geriatric fractures demonstrate intrinsic functional defects that drive impaired healing. Using flow cytometry, we analyzed and isolated hSSCs from callus tissue of five different skeletal sites (n=61) of patients ranging from 13 to 94years of age for functional and molecular studies. We observed that fracture-activated amplification of hSSC populations was comparable at all ages. However, functional analysis of isolated stem cells revealed that advanced age significantly correlated with reduced osteochondrogenic potential but was not associated with decreased in vitro clonogenicity. hSSCs derived from women displayed an exacerbated functional decline with age relative to those of aged men. Transcriptomic comparisons revealed downregulation of skeletogenic pathways such as WNT and upregulation of senescence-related pathways in young versus older hSSCs. Strikingly, loss of Sirtuin1 expression played a major role in hSSC dysfunction but re-activation by trans-resveratrol or a small molecule compound restored in vitro differentiation potential. These are the first findings that characterize age-related defects in purified hSSCs from geriatric fractures. Our results provide a foundation for future investigations into the mechanism and reversibility of skeletal stem cell aging in humans.
View details for DOI 10.1111/acel.13164
View details for PubMedID 32537886
Antibody Conditioning Enables MHC-Mismatched Hematopoietic Stem Cell Transplants and Organ Graft Tolerance.
Cell stem cell
Hematopoietic cell transplantation can correct hematological and immunological disorders by replacing a diseased blood system with a healthy one, but this currently requires depleting a patient's existing hematopoietic system with toxic and non-specific chemotherapy, radiation, or both. Here we report an antibody-based conditioning protocol with reduced toxicity and enhanced specificity for robust hematopoietic stem cell (HSC) transplantation and engraftment in recipient mice. Host pre-treatment with six monoclonal antibodies targeting CD47, Tcells, NK cells, and HSCs followed by donor HSC transplantation enabled stable hematopoietic system reconstitution in recipients with mismatches at half (haploidentical) or all major histocompatibility complex (MHC) genes. This approach allowed tolerance to heart tissue from HSC donor strains in haploidentical recipients, showing potential applications for solid organ transplantation without immune suppression. Fully mismatched chimeric mice developed antibody responses to nominal antigens, showing preserved functional immunity. These findings suggest approaches for transplanting immunologically mismatched HSCs and solid organs with limited toxicity.
View details for DOI 10.1016/j.stem.2019.05.018
View details for PubMedID 31204177
Identification of myosin II as a cripto binding protein and regulator of cripto function in stem cells and tissue regeneration.
Biochemical and biophysical research communications
Cripto regulates stem cell function in normal and disease contexts via TGFbeta/activin/nodal, PI3K/Akt, MAPK and Wnt signaling. Still, the molecular mechanisms that govern these pleiotropic functions of Cripto remain poorly understood. We performed an unbiased screen for novel Cripto binding proteins using proteomics-based methods, and identified novel proteins including members of myosin II complexes, the actin cytoskeleton, the cellular stress response, and extracellular exosomes. We report that myosin II, and upstream ROCK1/2 activities are required for localization of Cripto to cytoplasm/membrane domains and its subsequent release into the conditioned media fraction of cultured cells. Functionally, we demonstrate that soluble Cripto (one-eyed pinhead in zebrafish) promotes proliferation in mesenchymal stem cells (MSCs) and stem cell-mediated wound healing in the zebrafish caudal fin model of regeneration. Notably, we demonstrate that both Cripto and myosin II inhibitors attenuated regeneration to a similar degree and in a non-additive manner. Taken together, our data present a novel role for myosin II function in regulating subcellular Cripto localization and function in stem cells and an important regulatory mechanism of tissue regeneration. Importantly, these insights may further the development of context-dependent Cripto agonists and antagonists for therapeutic benefit.
View details for DOI 10.1016/j.bbrc.2018.12.059
View details for PubMedID 30579599