Integrated transcriptomics of human blood vessels defines a spatially controlled niche for early mesenchymal progenitor cells.

Human blood vessel walls show concentric layers, with the outermost tunica adventitia harboring mesenchymal progenitor cells. These progenitor cells maintain vessel homeostasis and provide a robust cell source for cell-based therapies. However, human adventitial stem cell niche has not been studied in detail. Here, using spatial and single-cell transcriptomics, we characterized the phenotype, potential, and microanatomic distribution of human perivascular progenitors. Initially, spatial transcriptomics identified heterogeneity between perivascular layers of arteries and veins and delineated the tunica adventitia into inner and outer layers. From this spatial atlas, we inferred a hierarchy of mesenchymal progenitors dictated by a more primitive cell with a high surface expression of CD201 (PROCR). When isolated from humans and mice, CD201Low expression typified a mesodermal committed subset with higher osteogenesis and less proliferation than CD201High cells, with a downstream effect on canonical Wnt signaling through DACT2. CD201Low cells also displayed high translational potential for bone tissue generation.

TrkA + sensory neurons regulate osteosarcoma proliferation and vascularization to promote disease progression.

Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multi-omics analyses of human OS bone samples and human dorsal root ganglia neurons further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. In order to curb tumor-associated axonal ingrowth, we next leveraged FDA-approved bupivacaine liposomes leading to significant reductions in sarcoma growth, vascularity, as well as alleviation of pain. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition appears to disrupt calcitonin receptor signaling (CALCR) and VEGF signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of osteosarcoma represent a novel adjunctive therapy to improve clinical outcomes and survival.

Requirement of Pdgfrα+ cells for calvarial bone repair.

Platelet-derived growth factor receptor α (PDGFRα) is often considered as a general marker of mesenchymal cells and fibroblasts, but also shows expression in a portion of osteoprogenitor cells. Within the skeleton, Pdgfrα+ mesenchymal cells have been identified in bone marrow and periosteum of long bones, where they play a crucial role in participating in fracture repair. A similar examination of Pdgfrα+ cells in calvarial bone healing has not been examined. Here, we utilize Pdgfrα-CreERTM;mT/mG reporter animals to examine the contribution of Pdgfrα+ mesenchymal cells to calvarial bone repair through histology and single-cell RNA sequencing (scRNA-Seq). Results showed that Pdgfrα+ mesenchymal cells are present in several cell clusters by scRNA-Seq, and by histology a dramatic increase in Pdgfrα+ cells populated the defect site at early timepoints to give rise to healed bone tissue overtime. Notably, diphtheria toxin-mediated ablation of Pdgfrα reporter+ cells resulted in significantly impaired calvarial bone healing. Our findings suggest that Pdgfrα-expressing cells within the calvarial niche play a critical role in the process of calvarial bone repair.

Mapping Somatosensory Afferent Circuitry to Bone Identifies Neurotrophic Signals Required for Fracture Healing.

The profound pain accompanying bone fracture is mediated by somatosensory neurons, which also appear to be required to initiate bone regeneration following fracture. Surprisingly, the precise neuroanatomical circuitry mediating skeletal nociception and regeneration remains incompletely understood. Here, we characterized somatosensory dorsal root ganglia (DRG) afferent neurons innervating murine long bones before and after experimental long bone fracture in mice. Retrograde labeling of DRG neurons by an adeno-associated virus with peripheral nerve tropism showed AAV-tdT signal. Single cell transcriptomic profiling of 6,648 DRG neurons showed highest labeling across CGRP+ neuron clusters (6.9-17.2%) belonging to unmyelinated C fibers, thinly myelinated Aδ fibers and Aß-Field LTMR (9.2%). Gene expression profiles of retrograde labeled DRG neurons over multiple timepoints following experimental stress fracture revealed dynamic changes in gene expression corresponding to the acute inflammatory ( S100a8 , S100a9 ) and mechanical force ( Piezo2 ). Reparative phase after fracture included morphogens such as Tgfb1, Fgf9 and Fgf18 . Two methods to surgically or genetically denervate fractured bones were used in combination with scRNA-seq to implicate defective mesenchymal cell proliferation and osteodifferentiation as underlying the poor bone repair capacity in the presence of attenuated innervation. Finally, multi-tissue scRNA-seq and interactome analyses implicated neuron-derived FGF9 as a potent regulator of fracture repair, a finding compatible with in vitro assessments of neuron-to-skeletal mesenchyme interactions.

Neuronal regulation of bone and tendon injury repair: a focused review.

Beyond the sensation of pain, peripheral nerves have been shown to play crucial roles in tissue regeneration and repair. As a highly innervated organ, bone can recover from injury without scar formation, making it an interesting model in which to study the role of nerves in tissue regeneration. As a comparison, tendon is a musculoskeletal tissue that is hypo-innervated, with repair often resulting in scar formation. Here, we reviewed the significance of innervation in three stages of injury repair (inflammatory, reparative, and remodeling) in two commonly injured musculoskeletal tissues: bone and tendon. Based on this focused review, we conclude that peripheral innervation is essential for phases of proper bone and tendon repair, and that nerves may dynamically regulate the repair process through interactions with the injury microenvironment via a variety of neuropeptides or neurotransmitters. A deeper understanding of neuronal regulation of musculoskeletal repair, and the crosstalk between nerves and the musculoskeletal system, will enable the development of future therapies for tissue healing.

Accumulating evidence has shown that, across organs systems, peripheral nerves regulate the process of tissue repair and regeneration. This is particularly relevant in the context of musculoskeletal injuries such as those affecting the bone and tendon. The question then arises: what is the function of peripheral innervation in the repair of bone and tendon injuries? This review offers an in-depth look at the ways in which nerves regulate the healing of bone and tendon injuries at various stages of recovery. A deeper comprehension of the influence of nerves on the repair of these tissues could pave the way for the development of future therapeutic strategies for tissue healing.

The Mohawk homeobox gene represents a marker and osteo-inhibitory factor in calvarial suture osteoprogenitor cells.

The regeneration of the mammalian skeleton's craniofacial bones necessitates the action of intrinsic and extrinsic inductive factors from multiple cell types, which function hierarchically and temporally to control the differentiation of osteogenic progenitors. Single-cell transcriptomics of developing mouse calvarial suture recently identified a suture mesenchymal progenitor population with previously unappreciated tendon- or ligament-associated gene expression profile. Here, we developed a Mohawk homeobox (MkxCG; R26RtdT) reporter mouse and demonstrated that this reporter identifies an adult calvarial suture resident cell population that gives rise to calvarial osteoblasts and osteocytes during homeostatic conditions. Single-cell RNA sequencing (scRNA-Seq) data reveal that Mkx+ suture cells display a progenitor-like phenotype with expression of teno-ligamentous genes. Bone injury with Mkx+ cell ablation showed delayed bone healing. Remarkably, Mkx gene played a critical role as an osteo-inhibitory factor in calvarial suture cells, as knockdown or knockout resulted in increased osteogenic differentiation. Localized deletion of Mkx in vivo also resulted in robustly increased calvarial defect repair. We further showed that mechanical stretch dynamically regulates Mkx expression, in turn regulating calvarial cell osteogenesis. Together, we define Mkx+ cells within the suture mesenchyme as a progenitor population for adult craniofacial bone repair, and Mkx acts as a mechanoresponsive gene to prevent osteogenic differentiation within the stem cell niche.

Vegfc-expressing cells form heterotopic bone after musculoskeletal injury.

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.

Neuronally differentiated macula densa cells regulate tissue remodeling and regeneration in the kidney.

Tissue regeneration is limited in several organs, including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest an existing remodeling capacity. This study uncovered endogenous tissue remodeling mechanisms in the kidney that were activated by the loss of body fluid and salt and regulated by a unique niche of a minority renal cell type called the macula densa (MD). Here, we identified neuronal differentiation features of MD cells that sense the local and systemic environment and secrete angiogenic, growth, and extracellular matrix remodeling factors, cytokines and chemokines, and control resident progenitor cells. Serial intravital imaging, MD nerve growth factor receptor and Wnt mouse models, and transcriptome analysis revealed cellular and molecular mechanisms of these MD functions. Human and therapeutic translation studies illustrated the clinical potential of MD factors, including CCN1, as a urinary biomarker and therapeutic target in chronic kidney disease. The concept that a neuronally differentiated key sensory and regulatory cell type responding to organ-specific physiological inputs controls local progenitors to remodel or repair tissues may be applicable to other organs and diverse tissue-regenerative therapeutic strategies.

Nr4a1 enhances Wnt4 transcription to promote mesenchymal stem cell osteogenesis and alleviates inflammation-inhibited bone regeneration.

Intense inflammatory response impairs bone marrow mesenchymal stem cell (BMSC)-mediated bone regeneration, with transforming growth factor (TGF)-ß1 being the most highly expressed cytokine. However, how to find effective and safe means to improve bone formation impaired by excessive TGF-ß1 remains unclear. In this study, we found that the expression of orphan nuclear receptor Nr4a1, an endogenous repressor of TGF-ß1, was suppressed directly by TGF-ß1-induced Smad3 and indirectly by Hdac4, respectively. Importantly, Nr4a1 overexpression promoted BMSC osteogenesis and reversed TGF-ß1-mediated osteogenic inhibition and pro-fibrotic effects. Transcriptomic and histologic analyses confirmed that upregulation of Nr4a1 increased the transcription of Wnt family member 4 (Wnt4) and activated Wnt pathway. Mechanistically, Nr4a1 bound to the promoter of Wnt4 and regulated its expression, thereby enhancing the osteogenic capacity of BMSCs. Moreover, treatment with Nr4a1 gene therapy or Nr4a1 agonist Csn-B could promote ectopic bone formation, defect repair, and fracture healing. Finally, we demonstrated the correlation of NR4A1 with osteogenesis and the activation of the WNT4/ß-catenin pathway in human BMSCs and fracture samples. Taken together, these findings uncover the critical role of Nr4a1 in bone formation and alleviation of inflammation-induced bone regeneration disorders, and suggest that Nr4a1 has the potential to be a therapeutic target for accelerating bone healing.

Isolation of Perivascular Mesenchymal Progenitor Cells from Human Adipose Tissue by Flow Cytometry.

Perivascular cells represent an in vivo counterpart of mesenchymal stromal/stem cells that populate the outer layer of blood vessels. Pericytes in capillaries and microvessels and adventitial cells of large arteries and veins give rise to stem/progenitor cells when isolated and cultured in vitro. These cells have been considered candidate cell types for cell therapy. Adipose tissue, being highly vascularized, dispensable, and easily accessed, is a viable option to obtain perivascular cells for use in research and in clinical trials. Here, we describe our established protocol to extract perivascular cells from human fat through fluorescence-activated cell sorting, which allows for the isolation of defined populations of progenitor cells with high reproducibility.

The HIF-1α/PLOD2 axis integrates extracellular matrix organization and cell metabolism leading to aberrant musculoskeletal repair.

While hypoxic signaling has been shown to play a role in many cellular processes, its role in metabolism-linked extracellular matrix (ECM) organization and downstream processes of cell fate after musculoskeletal injury remains to be determined. Heterotopic ossification (HO) is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues. Hypoxia and hypoxia-inducible factor 1α (HIF-1α) activation have been shown to promote HO. However, the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells (MPCs) contributes to pathologic bone formation remain to be elucidated. Here, we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analyses of the HO site, we found that collagen ECM organization is the most highly up-regulated biological process in MPCs. Zeugopod mesenchymal cell-specific deletion of Hif1α (Hoxa11-CreERT2; Hif1afl/fl) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreERT2; Hif1afl/fl mice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO. Importantly, our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion. From a translational aspect, a pan-LOX inhibitor significantly decreased HO. A newly screened compound revealed that the inhibition of PLOD2 activity in MPCs significantly decreased osteogenic differentiation and glycolytic metabolism. This suggests that the HIF-1α/PLOD2/LOX axis linked to metabolism regulates HO-forming MPC fate. These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promising strategy to mitigate HO formation.

The use of weakly supervised machine learning for necrosis assessment in patients with osteosarcoma: A pilot study.

Percent necrosis (PN) following chemotherapy is a prognostic factor for survival in osteosarcoma. Pathologists estimate PN by calculating tumor viability over an average of whole-slide images (WSIs). This non-standardized, labor-intensive process requires specialized training and has high interobserver variability. Therefore, we aimed to develop a machine-learning model capable of calculating PN in osteosarcoma with similar accuracy to that of a musculoskeletal pathologist. In this proof-of-concept study, we retrospectively obtained six WSIs from two patients with conventional osteosarcomas. A weakly supervised learning model was trained by using coarse and incomplete annotations of viable tumor, necrotic tumor, and nontumor tissue in WSIs. Weakly supervised learning refers to processes capable of creating predictive models on the basis of partially and imprecisely annotated data. Once "trained," the model segmented areas of tissue and determined PN of the same six WSIs. To assess model fidelity, the pathologist also estimated PN of each WSI, and we compared the estimates using Pearson's correlation and mean absolute error (MAE). MAE was 15% over the six samples, and 6.4% when an outlier was removed, for which the model inaccurately labeled cartilaginous tissue. The model and pathologist estimates were strongly, positively correlated (r = 0.85). Thus, we created and trained a weakly supervised machine learning model to segment viable tumor, necrotic tumor, and nontumor and to calculate PN with accuracy similar to that of a musculoskeletal pathologist. We expect improvement can be achieved by annotating cartilaginous and other mesenchymal tissue for better representation of the histological heterogeneity in osteosarcoma.

TrkA-mediated sensory innervation of injured mouse tendon supports tendon sheath progenitor cell expansion and tendon repair.

Peripheral neurons terminate at the surface of tendons partly to relay nociceptive pain signals; however, the role of peripheral nerves in tendon injury and repair remains unclear. Here, we show that after Achilles tendon injury in mice, there is new nerve growth near tendon cells that express nerve growth factor (NGF). Conditional deletion of the Ngf gene in either myeloid or mesenchymal mouse cells limited both innervation and tendon repair. Similarly, inhibition of the NGF receptor tropomyosin receptor kinase A (TrkA) abrogated tendon healing in mouse tendon injury. Sural nerve transection blocked the postinjury increase in tendon sensory innervation and the expansion of tendon sheath progenitor cells (TSPCs) expressing tubulin polymerization promoting protein family member 3. Single cell and spatial transcriptomics revealed that disruption of sensory innervation resulted in dysregulated inflammatory signaling and transforming growth factor-ß (TGFß) signaling in injured mouse tendon. Culture of mouse TSPCs with conditioned medium from dorsal root ganglia neuron further supported a role for neuronal mediators and TGFß signaling in TSPC proliferation. Transcriptomic and histologic analyses of injured human tendon biopsy samples supported a role for innervation and TGFß signaling in human tendon regeneration. Last, treating mice after tendon injury systemically with a small-molecule partial agonist of TrkA increased neurovascular response, TGFß signaling, TSPC expansion, and tendon tissue repair. Although further studies should investigate the potential effects of denervation on mechanical loading of tendon, our results suggest that peripheral innervation is critical for the regenerative response after acute tendon injury.

Heart rate recovery after orthostatic challenge and cardiopulmonary exercise testing in older individuals: prospective multicentre observational cohort study.

Background: Impaired vagal function in older individuals, quantified by the 'gold standard' delayed heart rate recovery after maximal exercise (HRRexercise), is an independent predictor of cardiorespiratory capacity and mortality (particularly when HRR ≤12 beats min-1). Heart rate also often declines after orthostatic challenge (HRRorthostatic), but the mechanism remains unclear. We tested whether HRRorthostatic reflects similar vagal autonomic characteristics as HRRexercise. Methods: Prospective multicentre cohort study of subjects scheduled for cardiopulmonary exercise testing (CPET) as part of routine care. Before undergoing CPET, heart rate was measured with participants seated for 3 min, before standing for 3 min (HRRorthostatic). HRRexercise 1 min after the end of CPET was recorded. The primary outcome was the correlation between mean heart rate change every 10 s for 1 min after peak heart rate was attained on standing and after exercise for each participant. Secondary outcomes were HRRorthostatic and peak VO2 compared between individuals with HRRexercise <12 beats min-1. Results: A total of 87 participants (mean age: 64 yr [95%CI: 61-66]; 48 (55%) females) completed both tests. Mean heart rate change every 10 s for 1 min after peak heart rate after standing and exercise was significantly correlated (R2=0.81; P<0.0001). HRRorthostatic was unchanged in individuals with HRRexercise ≤12 beats min-1 (n=27), but was lower when HRRexercise >12 beats min-1 (n=60; mean difference: 3 beats min-1 [95% confidence interval 1-5 beats min-1]; P<0.0001). Slower HRRorthostatic was associated with lower peak VO2 (mean difference: 3.7 ml kg-1 min-1 [95% confidence interval 0.7-6.8 ml kg-1 min-1]; P=0.039). Conclusion: Prognostically significant heart rate recovery after exhaustive exercise is characterised by quantitative differences in heart rate recovery after orthostatic challenge. These data suggest that orthostatic challenge is a valid, simple test indicating vagal impairment. Clinical trial registration: researchregistry6550.

Tppp3+ synovial/tendon sheath progenitor cells contribute to heterotopic bone after trauma.

Heterotopic ossification (HO) is a pathological process resulting in aberrant bone formation and often involves synovial lined tissues. During this process, mesenchymal progenitor cells undergo endochondral ossification. Nonetheless, the specific cell phenotypes and mechanisms driving this process are not well understood, in part due to the high degree of heterogeneity of the progenitor cells involved. Here, using a combination of lineage tracing and single-cell RNA sequencing (scRNA-seq), we investigated the extent to which synovial/tendon sheath progenitor cells contribute to heterotopic bone formation. For this purpose, Tppp3 (tubulin polymerization-promoting protein family member 3)-inducible reporter mice were used in combination with either Scx (Scleraxis) or Pdgfra (platelet derived growth factor receptor alpha) reporter mice. Both tendon injury- and arthroplasty-induced mouse experimental HO models were utilized. ScRNA-seq of tendon-associated traumatic HO suggested that Tppp3 is an early progenitor cell marker for either tendon or osteochondral cells. Upon HO induction, Tppp3 reporter+ cells expanded in number and partially contributed to cartilage and bone formation in either tendon- or joint-associated HO. In double reporter animals, both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells gave rise to HO-associated cartilage. Finally, analysis of human samples showed a substantial population of TPPP3-expressing cells overlapping with osteogenic markers in areas of heterotopic bone. Overall, these data demonstrate that synovial/tendon sheath progenitor cells undergo aberrant osteochondral differentiation and contribute to HO after trauma.

Differential pericyte marker expression in craniofacial benign and malignant vascular tumors.

BACKGROUND: Vascular anomalies and tumors are common in the head, neck, and craniofacial areas and are associated with abnormalities in the angiomatous architecture. However, the etiology and molecular basis for the pathogenesis of most vascular lesions are still unknown. Pericytes are mural cells that surround endothelial cells. Besides angiogenesis and other physiological functions, pericytes play an important role in vascularized tissue repair and as resident mesenchymal stem/progenitor cells. Perivascular cells demonstrate a distinct immunohistochemical profile, including expression of alpha-smooth muscle actin (α-SMA), CD146, CD105, and PDGFRß, without endothelial differentiation (absence of CD31 and CD34 immunoreactivity). These pericyte markers have been shown to be expressed in soft tissue hemangiomas. However, they have not been fully examined in intraosseous hemangiomas. METHODS: In this study, we compared mesenchymal stem cell (MSC) expression of CD146 and α-SMA markers in pericytes from hemangiomas from different tissues and malignant vascular tumors. RESULTS: The results demonstrated an increased expression of pericyte markers in perivascular cells of benign hemangiomas, especially intraosseous hemangiomas and a significantly reduced expression of pericyte markers in malignant angiosarcomas. CONCLUSION: The evidence provides insight into the function of pericytes in vascular tumors and suggests their role in vascular tumor disease types.

ZIC1 Dictates Osteogenesis Versus Adipogenesis in Human Mesenchymal Progenitor Cells Via a Hedgehog Dependent Mechanism.

Numerous intrinsic factors regulate mesenchymal progenitor commitment to a specific cell fate, such as osteogenic or adipogenic lineages. Identification and modulation of novel intrinsic regulatory factors represent an opportunity to harness the regenerative potential of mesenchymal progenitors. In the present study, the transcription factor (TF) ZIC1 was identified to be differentially expressed among adipose compared with skeletal-derived mesenchymal progenitor cells. We observed that ZIC1 overexpression in human mesenchymal progenitors promotes osteogenesis and prevents adipogenesis. ZIC1 knockdown demonstrated the converse effects on cell differentiation. ZIC1 misexpression was associated with altered Hedgehog signaling, and the Hedgehog antagonist cyclopamine reversed the osteo/adipogenic differentiation alterations associated with ZIC1 overexpression. Finally, human mesenchymal progenitor cells with or without ZIC1 overexpression were implanted in an ossicle assay in NOD-SCID gamma mice. ZIC1 overexpression led to significantly increased ossicle formation in comparison to the control, as assessed by radiographic and histologic measures. Together, these data suggest that ZIC1 represents a TF at the center of osteo/adipogenic cell fate determinations-findings that have relevance in the fields of stem cell biology and therapeutic regenerative medicine.

Aldehyde Dehydrogenase, a Marker of Normal and Malignant Stem Cells, Typifies Mesenchymal Progenitors in Perivascular Niches.

Innate mesenchymal stem cells exhibiting multilineage differentiation and tissue (re)generative-or pathogenic-properties reside in perivascular niches. Subsets of these progenitors are committed to either osteo-, adipo-, or fibrogenesis, suggesting the existence of a developmental organization in blood vessel walls. We evaluated herein the activity of aldehyde dehydrogenase, a family of enzymes catalyzing the oxidation of aldehydes into carboxylic acids and a reported biomarker of normal and malignant stem cells, within human adipose tissue perivascular areas. A progression of ALDHLow to ALDHHigh CD34+ cells was identified in the tunica adventitia. Mesenchymal stem cell potential was confined to ALDHHigh cells, as assessed by proliferation and multilineage differentiation in vitro of cells sorted by flow cytometry with a fluorescent ALDH substrate. RNA sequencing confirmed and validated that ALDHHigh cells have a progenitor cell phenotype and provided evidence that the main isoform in this fraction is ALDH1A1, which was confirmed by immunohistochemistry. This demonstrates that ALDH activity, which marks hematopoietic progenitors and stem cells in diverse malignant tumors, also typifies native, blood vessel resident mesenchymal stem cells.

TIAM1 acts as an actin organization regulator to control adipose tissue-derived pericyte cell fate.

Pericytes are multipotent mesenchymal precursor cells that demonstrate tissue-specific properties. In this study, by comparing human adipose tissue- and periosteum-derived pericyte microarrays, we identified T cell lymphoma invasion and metastasis 1 (TIAM1) as a key regulator of cell morphology and differentiation decisions. TIAM1 represented a tissue-specific determinant between predispositions for adipocytic versus osteoblastic differentiation in human adipose tissue-derived pericytes. TIAM1 overexpression promoted an adipogenic phenotype, whereas its downregulation amplified osteogenic differentiation. These results were replicated in vivo, in which TIAM1 misexpression altered bone or adipose tissue generation in an intramuscular xenograft animal model. Changes in pericyte differentiation potential induced by TIAM1 misexpression correlated with actin organization and altered cytoskeletal morphology. Small molecule inhibitors of either small GTPase Rac1 or RhoA/ROCK signaling reversed TIAM1-induced morphology and differentiation in pericytes. In summary, our results demonstrate that TIAM1 regulates the cellular morphology and differentiation potential of human pericytes, representing a molecular switch between osteogenic and adipogenic cell fates.