Preservation of ovarian function using human pluripotent stem cell-derived mesenchymal progenitor cells.

Ovarian reserve diminishes with age, and older women experience a corresponding shift in sex hormone levels. These changes contribute to an age-dependent decrease in fertility and a decline in overall health. Furthermore, while survival rates following cancer treatment have improved for young female patients, a reduction in ovarian function due to the side effects of such treatments can be difficult to avoid. To date, no effective therapy has been recommended to preserve ovarian health in these patients. Mesenchymal progenitor cells (MPCs) are considered a promising option for cell therapy aimed at maintaining fertility and fecundity. Although MPCs derived from human adult tissues are recognized for their various protective effects against ovarian senescence, they are limited in quantity. Consequently, human pluripotent stem cell-derived MPCs (hPSC-MPCs), which exhibit high proliferative capacity and retain genetic stability during growth, have been utilized to delay reproductive aging. This review highlights the impact of hPSC-MPCs on preserving the functionality of damaged ovaries in female mouse models subjected to chemotherapy and natural aging. It also proposes their potential as a valuable cell source for fertility preservation in women with a variety of diseases.

Three-dimensional texture analyses of multi-quantitative relaxation time maps for evaluating cartilage repair with the treatment of allogeneic human adipose-derived mesenchymal progenitor cells.

BACKGROUND: To explore the ability of three-dimensional texture analyses based on gray-level run-length matrix (GLRLM) for examining the spatial distribution of pixel values on magnetic resonance imaging (MRI) relaxation time maps and detecting the compositional variation of cartilage repair following treatment with allogeneic human adipose-derived mesenchymal progenitor cells (haMPCs). METHODS: Participants with knee osteoarthritis were randomly divided into three groups with intra-articular haMPCs injections: low-, medium-, and high-dose groups. We analyzed five GLRLM parameters in the T1rho, T2 and T2star maps, including run length non-uniformity (RLNonUni), gray-level non-uniformity (GLevNonU), long run emphasis (LngREmph), short run emphasis (ShrtREmp), and fraction of images in runs. We used the relative D values (the ratio of difference values to baseline) as the objective to avoid errors caused by individual differences. We calculated the two-tailed Pearson's linear correlation coefficient (r) to investigate the correlations of the texture parameters with the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores. RESULTS: Compared with the base time, significant reduction of WOMAC score was observed in both high and medium doses groups at terminal time, indicating relief of pain symptoms in high and medium groups with the treatment of allogeneic haMPCs. Significant differences were observed in the GLRLM parameters of cartilage MR relaxation time maps in different doses groups. In both T1rho and T2 relaxation time maps, the high-dose group showed significant increases in relative D values of RLNonUni, GLevNonU, LngREmph and ShrtREmp, which indicated significant changes in the uniformity of relaxation time maps. For T2star map, GLRLM parameters such as GLevNonU and ShrtREmp, especially LngREmph, showed significant increases in relative D values in high-dose group. Among all GLRLM features, LngREmph of three relaxation time maps had performed excellent linear correlations with WOMAC scores. CONCLUSIONS: Texture analysis of the cartilage may allow the detection of compositional variation in cartilage repair with the treatment of allogeneic haMPCs. This technique displays potential applications in understanding the mechanism of stem cell repair of the cartilage and assessing the treatment response.

Multiple treatments with human embryonic stem cell-derived mesenchymal progenitor cells preserved the fertility and ovarian function of perimenopausal mice undergoing natural aging.

OBJECTIVES: Currently, no approved stem cell-based therapies for preserving ovarian function during aging. To solve this problem, we developed a long-term treatment for human embryonic stem cell-derived mesenchymal progenitor cells (hESC-MPCs). We investigated whether the cells retained their ability to resist ovarian aging, which leads to delayed reproductive senescence. MATERIALS AND METHODS: In a middle-aged female model undergoing natural aging, we analyzed whether hESC-MPCs benefit the long-term maintenance of reproductive fecundity and ovarian reservoirs and how their transplantation regulates ovarian function. RESULTS: The number of primordial follicles and mice with regular estrous cycles were increased in perimenopausal mice who underwent multiple introductions of hESC-MPCs compared to age-matched controls. The estradiol levels in the hESC-MPCs group were restored to those in the young and adult groups. Embryonic development and live birth rates were higher in the hESC-MPC group than in the control group, suggesting that hESC-MPCs delayed ovarian senescence. In addition to their direct effects on the ovary, multiple-treatments with hESC-MPCs reduced ovarian fibrosis by downregulating inflammation and fibrosis-related genes via the suppression of myeloid-derived suppressor cells (MDSCs) produced in the bone marrow. CONCLUSIONS: Multiple introductions of hESC-MPCs could be a useful approach to prevent female reproductive senescence and that these cells are promising sources for cell therapy to postpone the ovarian aging and retain fecundity in perimenopausal women.

The Effects of Fucoidan Derived from Sargassum filipendula and Fucus vesiculosus on the Survival and Mineralisation of Osteogenic Progenitors.

Osteosarcoma is a bone cancer primarily affecting teenagers. It has a poor prognosis and diminished quality of life after treatment due to chemotherapy side effects, surgical complications and post-surgical osteoporosis risks. The sulphated polysaccharide fucoidan, derived from brown algae, has been a subject of interest for its potential anti-cancer properties and its impact on bone regeneration. This study explores the influence of crude, low-molecular-weight (LMW, 10-50 kDa), medium-molecular-weight (MMW, 50-100 kDa) and high-molecular-weight (HMW, >100 kDa) fractions from Sargassum filipendula, harvested from the Colombian sea coast, as well as crude fucoidan from Fucus vesiculosus, on a specific human osteoprogenitor cell type, human embryonic-derived mesenchymal stem cells. Fourier transform infrared spectroscopy coupled with attenuated total reflection (FTIR-ATR) results showed the highest sulphation levels and lowest uronic acid content in crude extract from F. vesiculosus. There was a dose-dependent drop in focal adhesion formation, proliferation and osteogenic differentiation of cells for all fucoidan types, but the least toxicity was observed for LMW and MMW. Transmission electron microscopy (TEM), JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimi-dazolylcarbocyanine iodide) staining and cytochrome c analyses confirmed mitochondrial damage, swollen ER and upregulated autophagy due to fucoidans, with the highest severity in the case of F. vesiculosus fucoidan. Stress-induced apoptosis-like cell death by F. vesiculosus fucoidan and stress-induced necrosis-like cell death by S. filipendula fucoidans were also confirmed. LMW and MMW doses of <200 ng/mL were the least toxic and showed potential osteoinductivity. This research underscores the multifaceted impact of fucoidans on osteoprogenitor cells and highlights the delicate balance between potential therapeutic benefits and the challenges involved in using fucoidans for post-surgery treatments in patients with osteosarcoma.

Brg1/PRMT5 nuclear complex epigenetically regulates FOXO1 in IPF mesenchymal progenitor cells.

We have discovered intrinsically fibrogenic mesenchymal progenitor cells (MPCs) in the human idiopathic pulmonary fibrosis (IPF) lung. IPF MPCs display a durably distinct transcriptome, suggesting that they have undergone epigenetic modifications. Prior studies indicate that the chromatin remodeler Brg1 associates with the arginine methyltransferase PRMT5 to epigenetically regulate transcription factors. We hypothesize that a Brg1/PRMT5 nuclear complex epigenetically regulates critical nodes in IPF MPC self-renewal signaling networks. IPF and control MPCs were isolated from primary mesenchymal cell lines established from IPF and control patients. RNA-sequencing identified increased expression of the FOXO1 transcription factor in IPF MPCs compared with controls, a result we confirmed by Q-PCR and Western blot analysis. Immunoprecipitation identified a CD44/Brg1/PRMT5 nuclear complex in IPF MPCs. Chromatin immunoprecipitation assays showed that PRMT5 and its methylation mark H3R2me2 are enriched on the FOXO1 promoter. We show that loss of Brg1 and PRMT5 function decreases FOXO1 expression and impairs IPF MPC self-renewal, and that loss of FOXO1 function decreases IPF MPC self-renewal and expression of the SOX2 and OCT4 stemness markers. Our findings indicate that the FOXO1 gene is overexpressed in IPF MPCs in a CD44/Brg1/PRMT5 nuclear complex-dependent manner. Our data suggest that Brg1 alters chromatin accessibility, enriching PRMT5 occupancy on the FOXO1 promoter, and PRMT5 methylates histone H3 arginine 2 (H3R2) on the FOXO1 promoter, increasing its expression. Our data are in accord with the concept that this coordinated interplay is responsible for promoting IPF MPC self-renewal and maintaining a critical pool of fibrogenic MPCs that drive IPF progression.NEW & NOTEWORTHY Our research offers valuable understanding regarding the epigenetic control of IPF MPC. The data we obtained strongly support the idea that the coordination between chromatin remodeling and histone methylation plays a key role in regulating transcription factors. Specifically, our findings indicate that FOXO1, an essential transcription factor, likely governs the self-renewal of IPF MPC, which is crucial for maintaining a critical pool of fibrogenic MPCs. This interplay could be an important therapeutic target.

Treatment of Naturally Occurring Tendon Disease with Allogeneic Multipotent Mesenchymal Stromal Cells: A Randomized, Controlled, Triple-Blinded Pilot Study in Horses.

The treatment of tendinopathies with multipotent mesenchymal stromal cells (MSCs) is a promising option in equine and human medicine. However, conclusive clinical evidence is lacking. The purpose of this study was to gain insight into clinical treatment efficacy and to identify suitable outcome measures for larger clinical studies. Fifteen horses with early naturally occurring tendon disease were assigned to intralesional treatment with allogeneic adipose-derived MSCs suspended in serum or with serum alone through block randomization (dosage adapted to lesion size). Clinicians and horse owners remained blinded to the treatment during 12 months (seven horses per group) and 18 months (seven MSC-group and five control-group horses) of follow-up including clinical examinations and diagnostic imaging. Clinical inflammation, lameness, and ultrasonography scores improved more over time in the MSC group. The lameness score difference significantly improved in the MSC group compared with the control group after 6 months. In the MSC group, five out of the seven horses were free of re-injuries and back to training until 12 and 18 months. In the control group, three out of the seven horses were free of re-injuries until 12 months. These results suggest that MSCs are effective for the treatment of early-phase tendon disease and provide a basis for a larger controlled study.

Hiding in Plain Sight: Human Gingival Fibroblasts as an Essential, Yet Overlooked, Tool in Regenerative Medicine.

Adult human gingival fibroblasts (HGFs), the most abundant cells in the oral cavity, are essential for maintaining oral homeostasis. Compared with other tissues, adult oral mucosal wounds heal regeneratively, without scarring. Relative to fibroblasts from other locations, HGFs are relatively refractory to myofibroblast differentiation, immunomodulatory, highly regenerative, readily obtained via minimally invasive procedures, easily and rapidly expanded in vitro, and highly responsive to growth factors and cytokines. Consequently, HGFs might be a superior, yet perhaps underappreciated, source of adult mesenchymal progenitor cells to use in tissue engineering and regeneration applications, including the treatment of fibrotic auto-immune connective tissue diseases such as scleroderma. Herein, we highlight in vitro and translational studies that have investigated the regenerative and differentiation potential of HGFs, with the objective of outlining current limitations and inspiring future research that could facilitate translating the regenerative potential of HGFs into the clinic.

Sustained notch signaling inhibition with a gamma-secretase inhibitor prevents traumatic heterotopic ossification.

Background: Traumatic heterotopic ossification (THO) is a devastating sequela following traumatic injuries and orthopedic surgeries. To date, the exact molecular mechanism of THO formation is still unclear, which hinders the development of effective treatments. The process of THO formation is believed to recapitulate a series of spatiotemporal cellular and signaling events that occur during skeletal development. The Notch signaling pathway is a critical genetic regulator in embryological bone development and fracture healing. However, few data are available concerning whether Notch signaling regulates THO development and maturation. Methods: We firstly detected the expressions of Notch target genes in both mouse and human THO samples with quantitative RT-PCR and immunohistochemistry (IHC). Then, tissue-resident mesenchymal progenitor cells (TMPCs) were isolated, and the abilities of the proliferation and osteogenic and chondrogenic differentiation of TMPCs were examined under the intervention of the gamma-secretase inhibitor-DAPT at different time points. Finally, DAPT was also administrated in THO mice by burn and Achilles tenotomy injury, and ectopic cartilage and bone formation were monitored by histology and micro-CT. Results: Several Notch target genes were upregulated in both mouse and human THO tissues. Sustained Notch signaling inhibition by DAPT reduced proliferation, osteogenic and chondrogenic differentiation of TMPCs in a time-dependent manner. Moreover, DAPT administration within 3 weeks could inhibit ectopic cartilage and bone formation in a mouse THO model without affecting the total body bone mass. Conclusions: The Notch signaling serves as an important therapeutic target during THO formation. And sustained gamma-secretase inhibition by DAPT has great potential in repressing chondrogenic and osteogenic differentiation of TMPCs, as well as inhibited ectopic cartilage and bone formation in vivo. The translational potential of this article: Sustained Notch inhibition via systemic DAPT (or other similar gamma-secretase inhibitors) administration has promising clinical utility for inhibiting THO formation, providing new insight into THO prophylaxis and treatment.

Analyzing the cellular and molecular atlas of ovarian mesenchymal cells provides a strategy against female reproductive aging.

Ovarian mesenchymal cells (oMCs) constitute a distinct microenvironment that supports folliculogenesis under physiological conditions. Supplementation of exogenous non-ovarian mesenchymal-related cells has been reported to be an efficient approach to improve ovarian functions. However, the development and cellular and molecular characteristics of endogenous oMCs remain largely unexplored. In this study, we surveyed the single-cell transcriptomic landscape to dissect the cellular and molecular changes associated with the aging of oMCs in mice. Our results showed that the oMCs were composed of five ovarian differentiated MC (odMC) populations and one ovarian mesenchymal progenitor (oMP) cell population. These cells could differentiate into various odMCs via an oMP-derived route to construct the ovarian stroma structures. Comparative analysis revealed that ovarian aging was associated with decreased quantity of oMP cells and reduced quality of odMCs. Based on the findings of bioinformatics analysis, we designed different strategies involving supplementation with young oMCs to examine their effects on female fertility and health. Our functional investigations revealed that oMCs supplementation prior to ovarian senescence was the optimal method to improve female fertility and extend the reproductive lifespan of aged females in the long-term.

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.

Mesenchymal progenitor cells from non-inflamed versus inflamed synovium post-ACL injury present with distinct phenotypes and cartilage regeneration capacity.

BACKGROUND: Osteoarthritis (OA) is a chronic debilitating disease impacting a significant percentage of the global population. While there are numerous surgical and non-invasive interventions that can postpone joint replacement, there are no current treatments which can reverse the joint damage occurring during the pathogenesis of the disease. While many groups are investigating the use of stem cell therapies in the treatment of OA, we still don't have a clear understanding of the role of these cells in the body, including heterogeneity of tissue resident adult mesenchymal progenitor cells (MPCs). METHODS: In the current study, we examined MPCs from the synovium and individuals with or without a traumatic knee joint injury and explored the chondrogenic differentiation capacity of these MPCs in vitro and in vivo. RESULTS: We found that there is heterogeneity of MPCs with the adult synovium and distinct sub-populations of MPCs and the abundancy of these sub-populations change with joint injury. Furthermore, only some of these sub-populations have the ability to effect cartilage repair in vivo. Using an unbiased proteomics approach, we were able to identify cell surface markers that identify this pro-chondrogenic MPC population in normal and injured joints, specifically CD82LowCD59+ synovial MPCs have robust cartilage regenerative properties in vivo. CONCLUSIONS: The results of this study clearly show that cells within the adult human joint can impact cartilage repair and that these sub-populations exist within joints that have undergone a traumatic joint injury. Therefore, these populations can be exploited for the treatment of cartilage injuries and OA in future clinical trials.

Overexpression of GREM1 Improves the Survival Capacity of Aged Cardiac Mesenchymal Progenitor Cells via Upregulation of the ERK/NRF2-Associated Antioxidant Signal Pathway.

Ischemic heart disease is the leading cause of mortality in the United States. Progenitor cell therapy can restore myocardial structure and function. However, its efficacy is severely limited by cell aging and senescence. Gremlin-1 (GREM1), a member of the bone morphogenetic protein antagonist family, has been implicated in cell proliferation and survival. However, GREM1's role in cell aging and senescence has never been investigated in human cardiac mesenchymal progenitor cells (hMPCs). Therefore, this study assessed the hypothesis that overexpression of GREM1 rejuvenates the cardiac regenerative potential of aging hMPCs to a youthful stage and therefore allows better capacity for myocardial repair. We recently reported that a subpopulation of hMPCs with low mitochondrial membrane potential can be sorted from right atrial appendage-derived cells in patients with cardiomyopathy and exhibit cardiac reparative capacity in a mouse model of myocardial infarction. In this study, lentiviral particles were used to overexpress GREM1 in these hMPCs. Protein and mRNA expression were assessed through Western blot and RT-qPCR. FACS analysis for Annexin V/PI staining and lactate dehydrogenase assay were used to assess cell survival. It was observed that cell aging and cell senescence led to a decrease in GREM1 expression. In addition, overexpression of GREM1 led to a decrease in expression of senescence genes. Overexpression of GREM1 led to no significant change in cell proliferation. However, GREM1 appeared to have an anti-apoptotic effect, with an increase in survival and decrease in cytotoxicity evident in GREM1-overexpressing hMPCs. Overexpressing GREM1 also induced cytoprotective properties by decreasing reactive oxidative species and mitochondrial membrane potential. This result was associated with increased expression of antioxidant proteins, such as SOD1 and catalase, and activation of the ERK/NRF2 survival signal pathway. Inhibition of ERK led to a decrease in GREM1-mediated rejuvenation in terms of cell survival, which suggests that an ERK-dependent pathway may be involved. Taken altogether, these results indicate that overexpression of GREM1 can allow aging hMPCs to adopt a more robust phenotype with improved survival capacity, which is associated with an activated ERK/NRF2 antioxidant signal pathway.

IL-8 concurrently promotes idiopathic pulmonary fibrosis mesenchymal progenitor cell senescence and PD-L1 expression enabling escape from immune cell surveillance.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease. We discovered fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients that display cell-autonomous fibrogenicity and drive fibrotic progression. In a study of the IPF MPC nuclear proteome, we identified DNA damage as one of the most altered functions in IPF MPCs. In prior work we found that IL-8 drives IPF MPC self-renewal. IL-8 can promote replicative stress and DNA damage and induce senescence through the CXCR2 receptor. We hypothesized that IL-8 promotes DNA damage-mediated senescence in IPF MPCs. We show that IL-8 induces DNA damage and promotes IPF MPC senescence. We discovered that IL-8 concurrently promotes senescence and upregulation of the programmed death ligand 1 (PD-L1) in a CXCR2-dependent manner. Disruption of programmed cell death protein-1 (PD-1)-PD-L1 interaction promotes natural killer (NK) cell killing of IPF MPCs in vitro and arrests IPF MPC-mediated experimental lung fibrosis in vivo. Immunohistochemical (IHC) analysis of IPF lung tissue identified PD-L1-expressing IPF MPCs codistributing with NK cells and ß-galactosidase-positive cells. Our data indicate that IL-8 simultaneously promotes IPF MPC DNA damage-induced senescence and high PD-L1 expression, enabling IPF MPCs to elude immune cell-targeted removal. Disruption of PD-1-PD-L1 interaction may limit IPF MPC-mediated fibrotic progression.NEW & NOTEWORTHY Here we show that IL-8 concurrently promotes senescence and upregulation of PD-L1 in IPF MPCs. IHC analysis identifies the presence of senescent IPF MPCs intermingled with NK cells in the fibroblastic focus, suggesting that senescent MPCs elude immune cell surveillance. We demonstrate that disruption of PD-1/PD-L1 interaction promotes NK cell killing of IPF MPCs and arrests IPF MPC-mediated experimental lung fibrosis. Disruption of PD-1/PD-L1 interaction may be one means to limit fibrotic progression.

A single-cell census of mouse limb development identifies complex spatiotemporal dynamics of skeleton formation.

Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naive limb progenitors and show that they differentiate progressively to form the skeleton in a complex, non-consecutive, three-dimensional pattern. Single-cell RNA sequencing of the developing mouse forelimb identified three progenitor states: naive, proximal, and autopodial, as well as Msx1 as a marker for the naive progenitors. In vivo lineage tracing confirmed this role and localized the naive progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1+ naive progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9+ chondroprogenitors, which occurs along all the forming skeletal segments. Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest an alternative model for limb skeleton development.

Ptip is essential for tooth development via regulating Wnt pathway.

OBJECTIVE: Epigenetic regulation plays important role in stem cell maintenance. Ptip was identified as epigenetic regulator, but the role in dental progenitor cells remains unclear. SUBJECTS AND METHODS: Dental mesenchymal progenitor cells were targeted by Sp7-icre and visualized in mTmG; Sp7-icre mice. The Ptipf/f ; Sp7-icre mice were generated and the phenotype of incisors and molars were shown by micro-computerized tomography, scanning electron microscope, hematoxylin & eosin staining, and immunofluorescence. Dental mesenchymal progenitor cells were sorted by fluorescence-activated cell sorting from lower incisors and RNA sequencing was performed. RESULTS: The Sp7-icre targets dental mesenchymal progenitor cells in incisors and molars. The Ptipf/f ; Sp7-icre mice showed spontaneous fractures in the cusp of upper incisors and lower incisors at 3 weeks (w), compensative overgrowth of lower incisors at 1 month (M), and overgrowth extended to the outside at 2 M. The molars showed shortened roots. The functions of odontoblasts and dental mesenchymal progenitor cells were impaired. Mechanically, loss of Ptip activates the Wnt pathway and upregulates the expression of Wls in dental mesenchymal progenitor cells. Also, the regenerative ability of lower incisors was significantly impaired. CONCLUSION: We first demonstrated that Ptip was crucial for tooth development via regulating Wnt signaling.

Molecular Characterization and Differentiation of Mesenchymal Progenitor Cells from Human Rheumatoid Arthritis Cartilage.

The presence of mesenchymal progenitor cells (MPCs) in rheumatoid arthritis (RA) articular cartilage is sparsely investigated largely owing to the persistent pathogenic disease condition and lack of specific biomarkers. Considering the recent advancements for potential cell-based therapies in immunomodulatory diseases, such as RA, this in vitro study was aimed at investigating the cellular, molecular, and differentiation characteristics of human RA cartilage-derived MPCs. Articular cartilage fragments from RA patients were obtained for the isolation of MPCs and characterization of their cellular and biological properties, cytogenetic stability, pluripotency, and plasticity. Established MPCs were phenotypically identified using a panel of markers, and their differentiation ability into mesenchymal lineages was assessed by cytochemical staining and the expression of molecular markers. MPCs displayed a heterogenous population of cells with characteristic features of multipotent stem cells. Cells had higher viability, proliferative rate, and colony-forming ability. Further, MPCs showed the expression of pluripotency markers, cytogenetic stability, and minimal replicative senescence. In addition, MPCs differentiated into osteocytes, adipocytes, and chondrocytes, and modulated the expression of each lineage-specific gene markers. The results demonstrated the availability of a viable pool of MPCs residing in RA cartilage, which could serve as an ideal cell source for reinstating native homotypic cartilage.

Prx1+ MPCs Accumulate in the Dura Mater of Wild-Type and p21-/- Mice Followed by a Specific Reduction in p21-/- Dural MPCs.

Epidural fat contains a population of mesenchymal progenitor cells (MPCs), and this study explores the behavior of these cells on the adjacent dura mater during growth and in response to injury in a p21 knockout mouse model. p21-/- mice are known to have increased cell proliferation and enhanced tissue regeneration post-injury. Therefore, it is hypothesized that the process by which epidural fat MPCs maintain the dura mater can be accelerated in p21-/- mice. Using a Prx1 lineage tracing mouse model, the epidural fat MPCs are found to increase in the dura mater over time in both C57BL/6 (p21+/+ ) and p21-/- mice; however, by 3 weeks post-tamoxifen induction, few MPCs are observed in p21-/- mice. These endogenous MPCs also localize to dural injuries in both mouse strains, with MPCs in p21-/- mice demonstrating increased proliferation. When epidural fat MPCs derived from p21-/- mice are transplanted into dural injuries in C57BL/6 mice, these MPCs are found in the injury site. It is demonstrated that epidural fat MPCs play a role in dural tissue maintenance and are able to directly contribute to dural injury repair. This suggests that these MPCs have the potential to treat injuries and/or pathologies in tissues surrounding the spinal cord.

Gene Expression and Chondrogenic Potential of Cartilage Cells: Osteoarthritis Grade Differences.

Recent data suggest that cells isolated from osteoarthritic (OA) cartilage express mesenchymal progenitor cell (MPC) markers that have the capacity to form hyaline-like cartilage tissue. Whether or not these cells are influenced by the severity of OA remains unexplored. Therefore, we analyzed MPC marker expression and chondrogenetic potential of cells from mild, moderate and severe OA tissue. Human osteoarthritic tibial plateaus were obtained from 25 patients undergoing total knee replacement. Each sample was classified as mild, moderate or severe OA according to OARSI scoring. mRNA expression levels of MPC markers-CD105, CD166, Notch 1, Sox9; mature chondrocyte markers-Aggrecan (Acan), Col II A1, hypertrophic chondrocyte and osteoarthritis-related markers-Col I A1, MMP-13 and ALPL were measured at the tissue level (day 0), after 2 weeks of in vitro expansion (day 14) and following chondrogenic in vitro re-differentiation (day 35). Pellet matrix composition after in vitro chondrogenesis of different OA-derived cells was tested for proteoglycans, collagen II and I by safranin O and immunofluorescence staining. Multiple MPC markers were found in OA cartilage resident tissue within a single OA joint with no significant difference between grades except for Notch1, which was higher in severe OA tissues. Expression levels of CD105 and Notch 1 were comparable between OA cartilage-derived cells of different disease grades and bone marrow mesenchymal stem cell (BM-MSC) line (healthy control). However, the MPC marker Sox 9 was conserved after in vitro expansion and significantly higher in OA cartilage-derived cells compared to its levels in the BM-MSC. The in vitro expansion of cartilage-derived cells resulted in enrichment while re-differentiation in reduction of MPC markers for all three analyzed grades. However, only moderate OA-derived cells after the in vitro chondrogenesis resulted in the formation of hyaline cartilage-like tissue. The latter tissue samples were also highly positive for collagen II and proteoglycans with no expression of osteoarthritis-related markers (collagen I, ALPL and MMP13). MPC marker expression did not differ between OA grades at the tissue level. Interestingly after in vitro re-differentiation, only moderate OA-derived cells showed the capacity to form hyaline cartilage-like tissue. These findings may have implications for clinical practice to understand the intrinsic repair capacity of articular cartilage in OA tissues and raises the possibility of these progenitor cells as a candidate for articular cartilage repair.

Single-cell mapping of regenerative and fibrotic healing responses after musculoskeletal injury.

After injury, a cascade of events repairs the damaged tissue, including expansion and differentiation of the progenitor pool and redeposition of matrix. To guide future wound regeneration strategies, we compared single-cell sequencing of regenerative (third phalangeal element [P3]) and fibrotic (second phalangeal element [P2]) digit tip amputation (DTA) models as well as traumatic heterotopic ossification (HO; aberrant). Analyses point to a common initial response to injury, including expansion of progenitors, redeposition of matrix, and activation of transforming growth factor ß (TGF-ß) and WNT pathways. Surprisingly, fibrotic P2 DTA showed greater transcriptional similarity to HO than to regenerative P3 DTA, suggesting that gene expression more strongly correlates with healing outcome than with injury type or cell origin. Differential analysis and immunostaining revealed altered activation of inflammatory pathways, such as the complement pathway, in the progenitor cells. These data suggests that common pathways are activated in response to damage but are fine tuned within each injury. Modulating these pathways may shift the balance toward regenerative outcomes.

Environmental oxygen affects ex vivo growth and proliferation of mesenchymal progenitors by modulating mitogen-activated protein kinase and mammalian target of rapamycin signaling.

BACKGROUND AIMS: Stem and progenitor cells of hematopoietic and mesenchymal lineages reside in the bone marrow under low oxygen (O2) saturation. O2 levels used in ex vivo expansion of multipotent mesenchymal stromal cells (MSCs) affect proliferation, metabolism and differentiation. METHODS: Using cell-based assays and transcriptome and proteome data, the authors compared MSC cultures simultaneously grown under a conventional 19.95% O2 atmosphere or at 5% O2. RESULTS: In 5% O2, MSCs showed better proliferation and higher self-renewal ability, most probably sustained by enhanced signaling activity of mitogen-activated protein kinase and mammalian target of rapamycin pathways. Non-oxidative glycolysis-based energy metabolism supported growth and proliferation in 5% O2 cultures, whereas MSCs grown under 19.95% O2 also utilized oxidative phosphorylation. Cytoprotection mechanisms used by cells under 5% O2 differed from 19.95% O2  suggesting differences in the triggers of cell stress between these two O2  conditions. CONCLUSIONS: Based on the potential benefits for the growth and metabolism of MSCs, the authors propose the use of 5% O2 for MSC culture.