Single-cell sequencing technology in diabetic wound healing:New insights into the progenitors-based repair strategies

Diabetes mellitus(DM),an increasingly prevalent chronic metabolic disease,is characterised by prolonged hyperglycaemia,which leads to long-term health consequences.Although much effort has been put into understanding the pathogenesis of diabetic wounds,the underlying mechanisms remain unclear.The advent of single-cell RNA sequencing(scRNAseq)has revolutionised biological research by enabling the identification of novel cell types,the discovery of cellular markers,the analysis of gene expression patterns and the prediction of develop-mental trajectories.This powerful tool allows for an in-depth exploration of pathogenesis at the cellular and molecular levels.In this editorial,we focus on progenitor-based repair strategies for diabetic wound healing as revealed by scRNAseq and highlight the biological behaviour of various healing-related cells and the alteration of signalling pathways in the process of diabetic wound healing.ScRNAseq could not only deepen our understanding of the complex biology of diabetic wounds but also identify and validate new targets for inter-vention,offering hope for improved patient outcomes in the management of this challenging complication of DM.

The characteristics of mRNA m^(6)A methylomes in allopolyploid Brassica napus and its diploid progenitors

Genome duplication events,comprising whole-genome duplication and single-gene duplication,produce a complex genomic context leading to multiple levels of genetic changes.However,the characteristics of m^(6)A modification,the most widespread internal eukaryotic mRNA modification,in polyploid species are still poorly understood.This study revealed the characteristics of m^(6)A methylomes within the early formation and following the evolution of allopolyploid Brassica napus.We found a complex relationship between m^(6)A modification abundance and gene expression level depending on the degree of enrichment or presence/absence of m^(6)A modification.Overall,the m^(6)A genes had lower gene expression levels than the non-m^(6)A genes.Allopolyploidization may change the expression divergence of duplicated gene pairs with identical m^(6)A patterns and diverged m^(6)A patterns.Compared with duplicated genes,singletons with a higher evolutionary rate exhibited higher m^(6)A modification.Five kinds of duplicated genes exhibited distinct distributions of m^(6)A modifications in transcripts and gene expression level.In particular,tandem duplication-derived genes showed unique m^(6)A modification enrichment around the transcript start site.Active histone modifications(H3K27ac and H3K4me3)but not DNA methylation were enriched around genes of m^(6)A peaks.These findings provide a new understanding of the features of m 6A modification and gene expression regulation in allopolyploid plants with sophisticated genomic architecture.

Soxllb regulates the migration and fate determination of Müller glia-derived progenitors during retina regeneration in zebrafish

The transcription factor Sox11 plays important roles in retinal neurogenesis during vertebrate eye development.However,its function in retina regeneration remains elusive.Here we report that Sox11 b,a zebrafish Sox11 homolog,regulates the migration and fate determination of Müller glia-derived progenitors(MGPCs)in an adult zebrafish model of mechanical retinal injury.Following a stab injury,the expression of Sox11 b was induced in proliferating MGPCs in the retina.Sox11 b knockdown did not affect MGPC formation at 4 days post-injury,although the nuclear morphology and subsequent radial migration of MGPCs were alte red.At 7 days post-injury,Sox11 b knockdown res ulted in an increased proportion of MGPCs in the inner retina and a decreased propo rtion of MGPCs in the outer nuclear layer,compared with controls.Furthermore,Sox11 b knockdown led to reduced photoreceptor regeneration,while it increased the numbe rs of newborn amacrines and retinal ganglion cells.Finally,quantitative polymerase chain reaction analysis revealed that Sox11 b regulated the expression of Notch signaling components in the retina,and Notch inhibition partially recapitulated the Sox11 b knockdown phenotype,indicating that Notch signaling functions downstream of Sox11 b.Our findings imply that Sox11 b plays key roles in MGPC migration and fate determination during retina regeneration in zebrafish,which may have critical im plications for future explorations of retinal repair in mammals.

Nutrient-regulated dynamics of chondroprogenitors in the postnatal murine growth plate

Longitudinal bone growth relies on endochondral ossification in the cartilaginous growth plate,where chondrocytes accumulate and synthesize the matrix scaffold that is replaced by bone.The chondroprogenitors in the resting zone maintain the continuous turnover of chondrocytes in the growth plate.Malnutrition is a leading cause of growth retardation in children;however,after recovery from nutrient deprivation,bone growth is accelerated beyond the normal rate,a phenomenon termed catch-up growth.Although nutritional status is a known regulator of long bone growth,it is largely unknown whether and how chondroprogenitor cells respond to deviations in nutrient availability.Here,using fate-mapping analysis in Axin2CreERT2 mice,we showed that dietary restriction increased the number of Axin2+chondroprogenitors in the resting zone and simultaneously inhibited their differentiation.Once nutrient deficiency was resolved,the accumulated chondroprogenitor cells immediately restarted differentiation and formed chondrocyte columns,contributing to accelerated growth.Furthermore,we showed that nutrient deprivation reduced the level of phosphorylated Akt in the resting zone and that exogenous IGF-1 restored the phosphorylated Akt level and stimulated differentiation of the pooled chondroprogenitors,decreasing their numbers.Our study of Axin2CreERT2 revealed that nutrient availability regulates the balance between accumulation and differentiation of chondroprogenitors in the growth plate and further demonstrated that IGF-1 partially mediates this regulation by promoting the committed differentiation of chondroprogenitor cells.

Transplanting neural progenitors to build a neuronal relay across the injured spinal cord

Cellular transplantation for repair of spinal cord injury is a prom- ising therapeutic strategy that includes the use of a variety of neural and non-neural cells isolated or derived from embryonic and adult tissue as well as embryonic stem cells and induced plu- ripotent stem cells. In particular, transplants of neural progenitor cells （NPCs） have been shown to limit secondary injury and scar formation and create a permissive environment in the injured spinal cord through the provision of neurotrophic molecules and growth supporting matrices that promote growth of injured host axons. Importantly, transplants of NPC are unique in their poten- tial to replace lost neural cells - including neurons, astrocytes,

Enrichment of retinal ganglion and Müller glia progenitors from retinal organoids derived from human induced pluripotent stem cells-possibilities and current limitations

BACKGROUND Retinal organoids serve as excellent human-specific disease models for conditions affecting otherwise inaccessible retinal tissue from patients.They permit the isolation of key cell types affected in various eye diseases including retinal ganglion cells(RGCs)and Müller glia.AIM To refine human-induced pluripotent stem cells(hiPSCs)differentiated into threedimensional(3D)retinal organoids to generate sufficient numbers of RGCs and Müller glia progenitors for downstream analyses.METHODS In this study we described,evaluated,and refined methods with which to generate Müller glia and RGC progenitors,isolated them via magnetic-activated cell sorting,and assessed their lineage stability after prolonged 2D culture.Putative progenitor populations were characterized via quantitative PCR and immunocytochemistry,and the ultrastructural composition of retinal organoid cells was investigated.RESULTS Our study confirms the feasibility of generating marker-characterized Müller glia and RGC progenitors within retinal organoids.Such retinal organoids can be dissociated and the Müller glia and RGC progenitor-like cells isolated via magnetic-activated cell sorting and propagated as monolayers.CONCLUSION Enrichment of Müller glia and RGC progenitors from retinal organoids is a feasible method with which to study cell type-specific disease phenotypes and to potentially generate specific retinal populations for cell replacement therapies.

Long-term and non-invasive in vivo tracking of DiD dye-labeled human hepatic progenitors in chronic liver disease models

BACKGROUND Chronic liver diseases(CLD)are the major public health burden due to the continuous increasing rate of global morbidity and mortality.The inherent limitations of organ transplantation have led to the development of stem cell-based therapy as a supportive and promising therapeutic option.However,identifying the fate of transplanted cells in vivo represents a crucial obstacle.AIM To evaluate the potential applicability of DiD dye as a cell labeling agent for longterm,and non-invasive in vivo tracking of transplanted cells in the liver.METHODS Magnetically sorted,epithelial cell adhesion molecule positive(1×106 cells/mL)fetal hepatic progenitor cells were labeled with DiD dye and transplanted into the livers of CLD-severe combined immunodeficiency(SCID)mice.Near-infrared(NIR)imaging was performed for in vivo tracking of the DiD-labeled transplanted cells along with colocalization of hepatic markers for up to 80 d.The existence of human cells within mouse livers was identified using Alu polymerase chain reaction and sequencing.RESULTS NIR fluorescence imaging of CLD-SCID mice showed a positive fluorescence signal of DiD at days 7,15,30,45,60,and 80 post-transplantation.Furthermore,positive staining of cytokeratin,c-Met,and albumin colocalizing with DiD fluorescence clearly demonstrated that the fluorescent signal of hepatic markers emerged from the DiD-labeled transplanted cells.Recovery of liver function was also observed with serum levels of glutamic-oxaloacetic transaminase,glutamate-pyruvate transaminase,and bilirubin.The detection of human-specific Alu sequence from the transplanted mouse livers provided evidence for the survival of transplanted cells at day 80.CONCLUSION DiD-labeling is promising for long-term and non-invasive in vivo cell tracking,and understanding the regenerative mechanisms incurred by the transplanted cells.

Production of Neural Progenitors from Bone Marrow Mesenchymal Stem Cells

In the brain, there are hundreds of types of specialized neurons and to generate one type of them we need to have neural progenitors for differentiation to specific neuron type. Mesenchymal stem cells (MSCs) are easily isolated, cultured, manipulated ex vivo, showing great potential for therapeutic applications. The adult MSCs have the potential to produce progeny that differentiate into a variety of cell types such as neurons. This fact suggests that MSCs derived neurons are an important cell type and a deep understanding of the molecular characteristics of it would significantly enhance the advancement of cell therapy for neurological disorders. Therefore, in this study, we isolated, identified, and studied neural progenitors by measuring expression levels through neurogenesis pathway of three neural differentiation markers nestin (NES), neurofilament (NF-L), and microtubule association protein (MAP-2) from mouse bone marrow MSCs (mouse bmMSCs) by using butylated hydroxyanisole (BHA) and diethyl sulfoxide (DMSO) as neural inducers agents. The results of immunocytochemistry and Real Time-PCR showed that in contrast to MSCs, neural differentiated cells showed neural progenitor pattern by showing stable increase in NES gene expression through differentiation process with increasing the protein expression through different exposures times, while NF-L gene and protein expression start to increased after 48 h but not replaced the NES expression completely even when its expression passed NES levels. The maturation marker Map-2 expression was low during the duration of differentiation period in protein and gene expression, which prove that these cells are still progenitors and can be redirected into specific type of neurons by further treatments.

Human adipose tissue contains erythroid progenitors expressing fetal hemoglobin

AIM: To investigate the origin of hematopoietic progenitors contained in the stromal vascular fraction(SVF)of human adipose tissue.METHODS: Tissue samples obtained from lipectomies were subjected to enzymatic digestion with collagenase to obtain a single-cell suspension. The centrifuged cell pellet, termed SVF, was separated immunomagnetically into CD45+and CD45-cells and cultured in serum-free medium containing hematopoietic cytokines. The freshly isolated and cultured cells were evaluated to determine their ability to form hematopoietic colony-forming units in clonogenic assays and for the expression of certain hematopoietic transcription factors by reversetranscription-polymerase chain reaction; the gene expression level was compared to that in CD34+hematopoietic progenitor cells from cord blood(CB) and adult peripheral blood(PB). To characterize erythroid progenitors, burst-forming units-erythroid(BFU-E) were developed in a semisolid medium under different culture conditions, and the hemoglobin composition and globin gene expression in the erythroid colonies were determined.RESULTS: The transcription factors SCL/TAL1, RUNX1,RUNX2 and GATA2 were expressed in both the CD45+and CD45-SVF populations; however, in contrast to our observations in the CD34+cells from CB and adult PB, GATA1 was not detected. Nevertheless, GATA1could be detected in the SVF cells after seven days in culture, whereas its expression was upregulated in the CB CD34+cells. The analysis of BFU-E-derived colonies revealed that virtually all erythroid cells produced by SVF cells expressed fetal hemoglobin, and the γ-globin mRNA levels ranged between those obtained in the adult- and neonatal-derived erythroid cells. Moreover,the SVF-derived erythroid cells synthesized similar levels of α- and β-globin mRNA, whereas the α-globin transcript levels were consistently higher those ofβ-globin in the cells derived from CB or PB CD34+cells. Furthermore, although the cellular distribution of hemoglobin in the erythroid cells derived from the CD34+cells obtained from hematopoietic tissues was dependent on the presence or absence of serum in the culture medium, this did not affect the SVF-derived erythroid cells.CONCLUSION: Our results demonstrate that hematopoietic progenitors in SVF have molecular and functional features that differ from those exhibited by circulating progenitors, suggesting the possibility of a different origin.

Multipotent pancreas progenitors: Inconclusive but pivotal topic

The establishment of multipotent pancreas progenitors(MPP) should have a significant impact not only on the ontology of the pancreas, but also for the translational research of glucose-responding endocrine b-cells. Deficiency of the latter may lead to the pandemic type 1 or type 2 diabetes mellitus, a metabolic disorder. An ideal treatment of which would potentially be the replacement of destroyed or failed b-cells, by restoring function of endogenous pancreatic endocrine cells or by transplantation of donor islets or in vitro generated insulin-secreting cells. Thus, considerable research efforts have been devoted to identify MPP candidates in the preand post-natal pancreas for the endogenous neogenesis or regeneration of endocrine insulin-secreting cells. In order to advance this inconclusive but critical field, we here review the emerging concepts, recent literature and newest developments of potential MPP and propose measures that would assist its forward progression.

Lineage restriction of adult human olfactory-derived progenitors to dopaminergic neurons

Human adult olfactory epithelium contains neural progenitors (hONPs) which replace damaged cellular components throughout life. Methods to isolate and expand the hONPs which form neurospheres in vitro have been developed in our laboratory. In response to morphogens, the hONPs differentiate along several neural lineages. This study optimized conditions for the differentiation of hONPs towards dopaminergic neurons. The hONPs were treated with Sonic hedgehog (Shh), in the presence or absence of retinoic acid (RA) and/or forskolin (FN). Transcription factors (nurr1, pitx3 and lmx1a) that promote embryonic mouse or chicken dopaminergic development were employed to determine if they would modulate lineage restriction of these adult human progenitors. Four expression vectors (pIRES-pitx3-nurr1, pLN-CX2-pitx3, pLNCX2-nurr1 and pLNCX2-lmx1a) were transfected into the hONPs, respectively. Transcription factor expression and the rate-limiting enzyme in dopamine synthesis tyrosine hydroxylase (TH) were detected in the transfected cells after 4 month-selection with G418, indicating transfected hONPs were stably restricted towards a dopaminergic lineage. Furthermore, a dopamine enzyme immunoassay (EIA) was employed to detect the synthesis and release of dopamine. The most efficient transfection paradigm was determined. Several neurotrophic factors were detected in the pre-transfected hONPs which have potential roles in the maintenance, survival and proliferation of dopaminergic neurons. Therefore the effect of transfection on the neurotrophin synthesis was examined. Transfection did not alter synthesis. The use of olfactory progenitors as a cell-based therapy for Parkinson’s disease (PD) would allow harvest without invasive surgery, provide an autologous cell population, eliminate need for immunosuppression and avoid the ethical concerns associated with embryonic tissues. This study suggests that specific transcription factors and treatment with morphogens can restrict human adult olfactory-derived progenitors to a dopaminergic neuronal lineage. Future studies will evaluate the utility of these unique cells in cell-replacement paradigms for the treatment of PD like animal models.

Unmasking the responses of the stem cells and progenitors in the subventricular zone after neonatal and pediatric brain injuries

There is great interest in the regenerative potential of the neural stem cells and progenitors that populate the subventricular zone（SVZ）. However, a comprehensive understanding of SVZ cell responses to brain injuries has been hindered by the lack of sensitive approaches to study the cellular composition of this niche. Here we review progress being made in deciphering the cells of the SVZ gleaned from the use of a recently designed flow cytometry panel that allows SVZ cells to be parsed into multiple subsets of progenitors as well as putative stem cells. We review how this approach has begun to unmask both the heterogeneity of SVZ cells as well as the dynamic shifts in cell populations with neonatal and pediatric brain injuries. We also discuss how flow cytometric analyses also have begun to reveal how specific cytokines, such as Leukemia inhibitory factor are coordinating SVZ responses to injury.

Spatially defined single-cell transcriptional profiling characterizes diverse chondrocyte subtypes and nucleus pulposus progenitors in human intervertebral discs

A comprehensive understanding of the cellular heterogeneity and molecular mechanisms underlying the development,homeostasis,and disease of human intervertebral disks(IVDs)remains challenging.Here,the transcriptomic landscape of 108108 IVD cells was mapped using single-cell RNA sequencing of three main compartments from young and adult healthy IVDs,including the nucleus pulposus(NP),annulus fibrosus,and cartilage endplate(CEP).The chondrocyte subclusters were classified based on their potential regulatory,homeostatic,and effector functions in extracellular matrix(ECM)homeostasis.Notably,in the NP,a PROCR+resident progenitor population showed enriched colony-forming unit-fibroblast(CFU-F)activity and trilineage differentiation capacity.Finally,intercellular crosstalk based on signaling network analysis uncovered that the PDGF and TGF-βcascades are important cues in the NP microenvironment.In conclusion,a single-cell transcriptomic atlas that resolves spatially regulated cellular heterogeneity together with the critical signaling that underlies homeostasis will help to establish new therapeutic strategies for IVD degeneration in the clinic.

Influence of endogenous ciliary neurotrophic factor on neural differentiation of adult rat hippocampal progenitors

Ciliary neurotrophic factor is the only known neurotrophic factor that can promote differentiation of hippocampal neural progenitor cells to glial cells and neurons in adult rats. This process is similar to spontaneous differentiation. Therefore, ciliary neurotrophic factor may be involved in spontaneous differentiation of neural stem cells. To verify this hypothesis, the present study isolated neural progenitor cells from adult male rats and cultured them in vitro. Results showed that when neural progenitor cells were cultured in the absence of mitogen fibroblast growth factor-2 or epidermal growth factor, they underwent spontaneous differentiation into neurons and glial cells. Western blot and immunocytochemical staining showed that exogenous ciliary neurotrophic factor strongly induced adult hippocampal progenitor cells to differentiate into neurons and glial cells. Moreover, passage 4 adult hippocampal progenitor cells expressed high levels of endogenous ciliary neurotrophic factor, and a neutralizing antibody against ciliary neurotrophic factor prevented the spontaneous neuronal and glial differentiation of adult hippocampal progenitor cells. These results suggest that the spontaneous differentiation of adult hippocampal progenitor cells is mediated partially by endogenous ciliary neurotrophic factor.

The collagen receptor,discoidin domain receptor 2,functions in Gli1-positive skeletal progenitors and chondrocytes to control bone development

Discoidin Domain Receptor 2(DDR2)is a collagen-activated receptor kinase that,together with integrins,is required for cells to respond to the extracellular matrix.Ddr2 loss-of-function mutations in humans and mice cause severe defects in skeletal growth and development.However,the cellular functions of Ddr2 in bone are not understood.Expression and lineage analysis showed selective expression of Ddr2 at early stages of bone formation in the resting zone and proliferating chondrocytes and periosteum.Consistent with these findings,Ddr2^(+)cells could differentiate into hypertrophic chondrocytes,osteoblasts,and osteocytes and showed a high degree of colocalization with the skeletal progenitor marker,Gli1.A conditional deletion approach showed a requirement for Ddr2 in Gli1-positive skeletal progenitors and chondrocytes but not mature osteoblasts.Furthermore,Ddr2 knockout in limb bud chondroprogenitors or purified marrow-derived skeletal progenitors inhibited chondrogenic or osteogenic differentiation,respectively.This work establishes a cell-autonomous function for Ddr2 in skeletal progenitors and cartilage and emphasizes the critical role of this collagen receptor in bone development.

Dorsal root ganglion progenitors differentiate to gamma-aminobutyric acid-and choline acetyltransferase-positive neurons

This study examined the isolation and differentiation of dorsal root ganglion progenitor cells for therapeutic use in neurodegenerative diseases. Rat embryonic dorsal root ganglia progenitors were isolated and purified using the differential adhesion method combined with cytosine arabinoside treatment. After culture in serum-free medium supplemented with B27, basic fibroblast growth factor and epidermal growth factor, these cells remained viable and survived for more than 18 months in vitro. Most cells differentiated to neurons that were immunoreactive for gamma-aminobutyric acid and choline acetyltransferase as detected by immunohistochemical staining. In addition, nerve growth factor and neurotrophic tyrosine kinase receptor expression were also observed in dorsal root ganglion progenitors and differentiated cells. K252a, an inhibitor that blocks nerve growth factor-induced signaling, inhibited cell survival, suggesting the possible existence of a nerve growth factor autocrine loop in these proliferating cells.

Renal progenitors: Roles in kidney disease and regeneration

Kidney disease is a devastating condition that affects millions of people worldwide, and its prevalence predicted to significantly increase. The kidney is complex organ encompassing many diverse cell type organized in a elaborate tissue architecture, makin regeneration a challenging feat. In recent years, there ha been a surge in the field of stem cell research to develo regenerative therapies for various organ systems. Here we review some recent progressions in characterizing th role of renal progenitors in development, regeneration and kidney disease in mammals. We also discuss how the zebrafish provides a unique experimental anima model that can provide a greater molecular and genet understanding of renal progenitors, which may contribut to the development of potential regenerative therapies fo human renal afflictions.

Identification and differentiation of PDX1 β-cell progenitors within the human pancreatic epithelium

AIM:To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm thatβ-cell progenitors reside within the pancreatic epithelium.METHODS:Due to mesenchymal stem cell(MSC)expansion and overgrowth,progenitor cells within the pancreatic epithelium cannot be characterized in vitro,thoughβ-cell dedifferentiation and expansion of MSC intermediates via epithelial-mesenchymal transition(EMT)may generateβ-cell progenitors.Pancreatic epithelial cells from endocrine and non-endocrine tissue were expanded and differentiated in a novel pancreatic epithelial expansion medium supplemented with growth factors known to support epithelial cell growth(dexamethasone,epidermal growth factor,3,5,3’-triiodo-l-thyronine,bovine brain extract).Cells were also infected with a single and dual lentiviral reporter prior to cell differentiation.Enhanced green fluorescent protein was controlled by the rat Insulin 1 promoter and the monomeric red fluorescent protein was controlled by the mouse PDX1 promoter.In combination with lentiviral tracing,cells expanded and differentiated in the pancreatic medium were characterized by flow cytometry(BD fluorescence activated cell sorting),immunostaining and real-time polymerase chain reaction(PCR)(7900HT Fast Realtime PCR System).RESULTS:In the presence of 10%serum MSCs rapidly expand in vitro while the epithelial cell population declines.The percentage of vimentin+cells increased from 22%±5.83%to 80.43%±3.24%(14 d)and99.00%±0.0%(21 d),and the percentage of epithelial cells decreased from 74.71%±8.34%to 26.57%±9.75%(14 d)and 4.00%±1.53%(21 d),P<0.01 for all time points.Our novel pancreatic epithelial expansion medium preserved the epithelial cell phenotype and minimized epithelial cell dedifferentiation and EMT.Cells expanded in our epithelial medium contained significantly less mesenchymal cells(vimentin+)compared to controls(44.87%±4.93%vs 95.67%±1.36%;P<0.01).During cell differentiation lentiviral reporting demonstrated that,PDX1+and insulin+cells were localized within adherent epithelial cell aggregates compared to controls.Compared to starting islets differentiated cells had at least two fold higher gene expression of PDX1,insulin,PAX4 and RFX(P<0.05).CONCLUSION:PDX1+cells were confined to adherent epithelial cell aggregates and not vimentin+cells(mesenchymal),suggesting that EMT is not a mechanism for generating pancreatic progenitor cells.

Type Ⅱ collagen-positive progenitors are important stem cells in controlling skeletal development and vascular formation

Type II collagen-positive(Col2^(+))cells have been reported as skeletal stem cells(SSCs),but the contribution of Col2^(+)progenitors to skeletal development both prenatally and postnatally during aging remains unclear.To address this question,we generated new mouse models with ablation of Col2^(+)cells at either the embryonic or postnatal stages.The embryonic ablation of Col2^(+)progenitors resulted in the death of newborn mice due to a decrease in skeletal blood vessels,loss of all vertebral bones and absence of most other bones except part of the craniofacial bone,the clavicle bone and a small piece of the long bone and ribs,which suggested that intramembranous ossification is involved in long bone development but does not participate in spine development.The postnatal ablation of Col2^(+)cells resulted in mouse growth retardation and a collagenopathy phenotype.Lineage tracing experiments with embryonic or postnatal mice revealed that Col2^(+)progenitors occurred predominantly in the growth plate(GP)and articular cartilage,but a limited number of Col2^(+)cells were detected in the bone marrow.Moreover,the number and differentiation ability of Col2^(+)progenitors in the long bone and knee joints decreased with increasing age.The fate-mapping study further revealed Col2^(+)lineage cells contributed to,in addition to osteoblasts and chondrocytes,CD31^(+)blood vessels in both the calvarial bone and long bone.Specifically,almost all blood vessels in calvarial bone and 25.4%of blood vessels in long bone were Col2^(+)lineage cells.However,during fracture healing,95.5%of CD31^(+)blood vessels in long bone were Col2^(+)lineage cells.In vitro studies further confirmed that Col2^(+)progenitors from calvarial bone and GP could form CD31^(+)vascular lumens.Thus,this study provides the first demonstration that intramembranous ossification is involved in long bone and rib development but not spine development.Col2^(+)progenitors contribute to CD31^(+)skeletal blood vessel formation,but the percentage differs between long bone and skull bone.The number and differentiation ability of Col2^(+)progenitors decreases with increasing age.

Spinal cord injury reprograms muscle fibroadipogenic progenitors to form heterotopic bones within muscles

The cells of origin of neurogenic heterotopic ossifications(NHOs), which develop frequently in the periarticular muscles following spinal cord injuries(SCIs) and traumatic brain injuries, remain unclear because skeletal muscle harbors two progenitor cell populations: satellite cells(SCs), which are myogenic, and fibroadipogenic progenitors(FAPs), which are mesenchymal. Lineage-tracing experiments using the Cre recombinase/Lox P system were performed in two mouse strains with the fluorescent protein Zs Green specifically expressed in either SCs or FAPs in skeletal muscles under the control of the Pax7 or Prrx1 gene promoter, respectively. These experiments demonstrate that following muscle injury, SCI causes the upregulation of PDGFRα expression on FAPs but not SCs and the failure of SCs to regenerate myofibers in the injured muscle, with reduced apoptosis and continued proliferation of muscle resident FAPs enabling their osteogenic differentiation into NHOs. No cells expressing Zs Green under the Prrx1 promoter were detected in the blood after injury, suggesting that the cells of origin of NHOs are locally derived from the injured muscle. We validated these findings using human NHO biopsies. PDGFRα+mesenchymal cells isolated from the muscle surrounding NHO biopsies could develop ectopic human bones when transplanted into immunocompromised mice, whereas CD56+myogenic cells had a much lower potential. Therefore, NHO is a pathology of the injured muscle in which SCI reprograms FAPs to undergo uncontrolled proliferation and differentiation into osteoblasts.