Influence of donor age and comorbidities on transduced human adipose-derived stem cell in vitro osteogenic potential.

Human adipose-derived mesenchymal stem cells (ASCs) transduced with a lentiviral vector system to express bone morphogenetic protein 2 (LV-BMP-2) have been shown to reliably heal bone defects in animal models. However, the influence of donor characteristics such as age, sex, race, and medical co-morbidities on ASC yield, growth and bone regenerative capacity, while critical to the successful clinical translation of stem cell-based therapies, are not well understood. Human ASCs isolated from the infrapatellar fat pads in 122 ASC donors were evaluated for cell growth characteristics; 44 underwent additional analyses to evaluate in vitro osteogenic potential, with and without LV-BMP-2 transduction. We found that while female donors demonstrated significantly higher cell yield and ASC growth rates, age, race, and the presence of co-morbid conditions were not associated with differences in proliferation. Donor demographics or the presence of comorbidities were not associated with differences in in vitro osteogenic potential or stem cell differentiation, except that transduced ASCs from healthy donors produced more BMP-2 at day 2. Overall, donor age, sex, race, and the presence of co-morbid conditions had a limited influence on cell yield, proliferation, self-renewal capacity, and osteogenic potential for non-transduced and transduced (LV-BMP-2) ASCs. These results suggest that ASCs are a promising resource for both autologous and allogeneic cell-based gene therapy applications.

Drug-induced modulation of gp130 signalling prevents articular cartilage degeneration and promotes repair.

OBJECTIVE: Human adult articular cartilage (AC) has little capacity for repair, and joint surface injuries often result in osteoarthritis (OA), characterised by loss of matrix, hypertrophy and chondrocyte apoptosis. Inflammation mediated by interleukin (IL)-6 family cytokines has been identified as a critical driver of proarthritic changes in mouse and human joints, resulting in a feed-forward process driving expression of matrix degrading enzymes and IL-6 itself. Here we show that signalling through glycoprotein 130 (gp130), the common receptor for IL-6 family cytokines, can have both context-specific and cytokine-specific effects on articular chondrocytes and that a small molecule gp130 modulator can bias signalling towards anti-inflammatory and antidegenerative outputs. METHODS: High throughput screening of 170 000 compounds identified a small molecule gp130 modulator termed regulator of cartilage growth and differentiation (RCGD 423) that promotes atypical homodimeric signalling in the absence of cytokine ligands, driving transient increases in MYC and pSTAT3 while suppressing oncostatin M- and IL-6-mediated activation of ERK and NF-κB via direct competition for gp130 occupancy. RESULTS: This small molecule increased proliferation while reducing apoptosis and hypertrophic responses in adult chondrocytes in vitro. In a rat partial meniscectomy model, RCGD 423 greatly reduced chondrocyte hypertrophy, loss and degeneration while increasing chondrocyte proliferation beyond that observed in response to injury. Moreover, RCGD 423 improved cartilage healing in a rat full-thickness osteochondral defect model, increasing proliferation of mesenchymal cells in the defect and also inhibiting breakdown of cartilage matrix in de novo generated cartilage. CONCLUSION: These results identify a novel strategy for AC remediation via small molecule-mediated modulation of gp130 signalling.

The Wnt5a Receptor, Receptor Tyrosine Kinase-Like Orphan Receptor 2, Is a Predictive Cell Surface Marker of Human Mesenchymal Stem Cells with an Enhanced Capacity for Chondrogenic Differentiation.

Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase-like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2-ve or unfractionated MSCs. In a sheep cartilage-repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing human cartilage, adult bone marrow, and adipose tissue. Their frequency in bone marrow was significantly lower in patients with osteoarthritis (OA) than in controls. However, after isolation of these cells and their initial expansion in vitro, there was greater ROR2 expression in the population derived from OA patients compared with controls. Furthermore, osteoarthritis-derived MSCs were better able to form cartilage than MSCs from control patients in a tissue engineering assay. We conclude that MSCs expressing high levels of ROR2 provide a defined population capable of predictably enhanced cartilage production. Stem Cells 2017;35:2280-2291.

Genetic Tagging During Human Mesoderm Differentiation Reveals Tripotent Lateral Plate Mesodermal Progenitors.

Although clonal studies of lineage potential have been extensively applied to organ specific stem and progenitor cells, much less is known about the clonal origins of lineages formed from the germ layers in early embryogenesis. We applied lentiviral tagging followed by vector integration site analysis (VISA) with high-throughput sequencing to investigate the ontogeny of the hematopoietic, endothelial and mesenchymal lineages as they emerge from human embryonic mesoderm. In contrast to studies that have used VISA to track differentiation of self-renewing stem cell clones that amplify significantly over time, we focused on a population of progenitor clones with limited self-renewal capability. Our analyses uncovered the critical influence of sampling on the interpretation of lentiviral tag sharing, particularly among complex populations with minimal clonal duplication. By applying a quantitative framework to estimate the degree of undersampling we revealed the existence of tripotent mesodermal progenitors derived from pluripotent stem cells, and the subsequent bifurcation of their differentiation into bipotent endothelial/hematopoietic or endothelial/mesenchymal progenitors. Stem Cells 2016;34:1239-1250.

Parylene scaffold for cartilage lesion.

Evaluate parylene scaffold feasibility in cartilage lesion treatment, introducing a novel paradigm combining a reparative and superficial reconstructive procedure. Fifteen rabbits were used. All animals had both knees operated and the same osteochondral lesion model was created bilaterally. The parylene scaffold was implanted in the right knee, and the left knee of the same animal was used as control. The animals were euthanized at different time points after surgery: four animals at three weeks, three animals at six weeks, four animals at nine weeks, and four animals at 12 weeks. Specimens were analyzed by International Cartilage Repair Society (ICRS) macroscopic evaluation, modified Pineda histologic evaluation of cartilage repair, and collagen II immunostaining. Parylene knees were compared to its matched contra-lateral control knees of the same animal using the Wilcoxon matched-pairs signed rank. ICRS mean ± SD values for parylene versus control, three, six, nine and twelve weeks, respectively: 7.83 ± 1.85 versus 4.42 ± 1.08, p = 0.0005; 10.17 ± 1.17 versus 6.83 ± 1.17, p = 0.03; 10.89 ± 0.60 versus 7.33 ± 2.18, p = 0.007; 10.67 ± 0.78 versus 7.83 ± 3.40, p = 0.03. Modified Pineda mean ± SD values for parylene versus control, six, nine and twelve weeks, respectively: 3.37 ± 0.87 versus 6.94 ± 1.7, p < 0.0001; 5.73 ± 2.05 versus 6.41 ± 1.7, p = 0.007; 3.06 ± 1.61 versus 6.52 ± 1.51, p < 0.0001. No inflammation was seen. Parylene implanted knees demonstrated higher collagen II expression via immunostaining in comparison to the control knees. Parylene scaffolds are a feasible option for cartilage lesion treatment and the combination of a reparative to a superficial reconstructive procedure using parylene scaffolds led to better results than the reparative procedure alone.

Right-Left Differences in Knee Extension Stiffness for the Normal Rat Knee: In Vitro Measurements Using a New Testing Apparatus.

Knee stiffness following joint injury or immobilization is a common clinical problem, and the rat has been used as a model for studies related to joint stiffness and limitation of motion. Knee stiffness measurements have been reported for the anesthetized rat, but it is difficult to separate the contributions of muscular and ligamentous restraints to the recorded values. in vitro testing of isolated rat knees devoid of musculature allows measurement of joint structural properties alone. In order to measure the effects of therapeutic or surgical interventions designed to alter joint stiffness, the opposite extremity is often used as a control. However, right-left stiffness differences for the normal rat knee have not been reported in the literature. If stiffness changes observed for a treatment group are within the normal right-left variation, validity of the results could be questioned. The objectives of this study were to utilize a new testing apparatus to measure right-left stiffness differences during knee extension in a population of normal rat knees and to document repeatability of the stiffness measurements on successive testing days. Moment versus rotation curves were recorded for 15 right-left pairs of normal rat knees on three consecutive days, with overnight specimen storage in a refrigerator. Each knee was subjected to ten loading-unloading cycles, with the last loading curve used for analysis. Angular rotation (AR), defined here as the change in flexion-extension angle from a specified applied joint moment, is commonly used as a measure of overall joint stiffness. For these tests, ARs were measured from the recorded test curves with a maximum applied extension moment of 100 g cm. Mean rotations for testing days 2 and 3 were 0.81-1.25 deg lower (p < 0.001) than for day 1, but were not significantly different from each other. For each testing day, mean rotations for right knees were 1.12-1.30 deg greater (p < 0.001) than left knees. These right-left stiffness differences should be considered when interpreting the results of knee treatment studies designed to alter knee stiffness when using the opposite extremity as a control.

Platelets contain tissue factor pathway inhibitor-2 derived from megakaryocytes and inhibits fibrinolysis.

Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pM) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with K(d) ~9 nM and binds FVa with K(d) ~100 nM. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with K(d) ~36-48 nM and bind FVa with K(d) ~252-456 nM. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (~7 nM) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis.

Perivascular support of human hematopoietic stem/progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) emerge and develop adjacent to blood vessel walls in the yolk sac, aorta-gonad-mesonephros region, embryonic liver, and fetal bone marrow. In adult mouse bone marrow, perivascular cells shape a "niche" for HSPCs. Mesenchymal stem/stromal cells (MSCs), which support hematopoiesis in culture, are themselves derived in part from perivascular cells. In order to define their direct role in hematopoiesis, we tested the ability of purified human CD146(+) perivascular cells, as compared with unfractionated MSCs and CD146(-) cells, to sustain human HSPCs in coculture. CD146(+) perivascular cells support the long-term persistence, through cell-to-cell contact and at least partly via Notch activation, of human myelolymphoid HSPCs able to engraft primary and secondary immunodeficient mice. Conversely, unfractionated MSCs and CD146(-) cells induce differentiation and compromise ex vivo maintenance of HSPCs. Moreover, CD146(+) perivascular cells express, natively and in culture, molecular markers of the vascular hematopoietic niche. Unexpectedly, this dramatic, previously undocumented ability to support hematopoietic stem cells is present in CD146(+) perivascular cells extracted from the nonhematopoietic adipose tissue.

Inactivation of a non-canonical gp130 signaling arm attenuates chronic systemic inflammation and multimorbidity induced by a high-fat diet.

Interleukin-6 (IL-6) is a major pro-inflammatory cytokine for which the levels in plasma demonstrate a robust correlation with age and body mass index (BMI) as part of the senescence-associated secretory phenotype. IL-6 cytokines also play a crucial role in metabolic homeostasis and regenerative processes, primarily via the canonical STAT3 pathway. Thus, selective modulation of IL-6 signaling may offer a unique opportunity for therapeutic interventions. Recently, we discovered that a non-canonical signaling pathway downstream of tyrosine (Y) 814 within the intracellular domain of gp130, the IL-6 co-receptor, is responsible for the recruitment and activation of SRC family of kinases (SFK). Mice with constitutive genetic inactivation of gp130 Y814 (F814 mice) show accelerated resolution of inflammatory response and superior regenerative outcomes in skin wound healing and posttraumatic models of osteoarthritis. The current study was designed to explore if selective genetic or pharmacological inhibition of the non-canonical gp130-Y814/SFK signaling reduces systemic chronic inflammation and multimorbidity in a high-fat diet (HFD)-induced model of accelerated aging. F814 mice showed significantly reduced inflammatory response to HFD in adipose and liver tissue, with significantly reduced levels of systemic inflammation compared to wild type mice. F814 mice were also protected from HFD-induced bone loss and cartilage degeneration. Pharmacological inhibition of gp130-Y814/SFK in mice on HFD mirrored the effects observed in F814 mice on HFD; furthermore, this pharmacological treatment also demonstrated a marked increase in physical activity levels and protective effects against inflammation-associated suppression of neurogenesis in the brain tissue compared to the control group. These findings suggest that selective inhibition of SFK signaling downstream of gp130 receptor represents a promising strategy to alleviate systemic chronic inflammation. Increased degenerative changes and tissue senescence are inevitable in obese and aged organisms, but we demonstrated that the systemic response and inflammation-associated multi-morbidity can be therapeutically mitigated.

Novel aspects of parenchymal-mesenchymal interactions: from cell types to molecules and beyond.

Mesenchymal stem or stromal cells (MSCs) were initially isolated from the bone marrow and received their name on the basis of their ability to differentiate into multiple lineages such as bone, cartilage, fat and muscle. However, more recent studies suggest that MSCs residing in perivascular compartments of the small and large blood vessels play a regulatory function supporting physiologic and pathologic responses of parenchymal cells, which define the functional representation of an organ or tissue. MSCs secrete or express factors that reach neighbouring parenchymal cells via either a paracrine effect or a direct cell-to-cell interaction promoting functional activity, survival and proliferation of the parenchymal cells. Previous concept of 'epithelial-stromal' interactions can now be widened. Given that MSC can also support hematopoietic, neuronal and other non-epithelial parenchymal lineages, terms 'parenchymal-stromal' or 'parenchymal-mesenchymal' interactions may better describe the supportive or 'trophic' functions of MSC. Importantly, in many cases, MSCs specifically provide supportive microenvironment for the most primitive stem or progenitor populations and therefore can play a role as 'stem/progenitor niche' forming cells. So far, regulatory roles of MSCs have been reported in many tissues. In this review article, we summarize the latest studies that focused on the supportive function of MSC. This thread of research leads to a new perspective on the interactions between parenchymal and mesenchymal cells and justifies a principally novel approach for regenerative medicine based on co-application of MSC and parenchymal cell for the most efficient tissue repair.

Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells.

Our understanding of how mesodermal tissue is formed has been limited by the absence of specific and reliable markers of early mesoderm commitment. We report that mesoderm commitment from human embryonic stem cells (hESCs) is initiated by epithelial-to-mesenchymal transition (EMT) as shown by gene expression profiling and by reciprocal changes in expression of the cell surface proteins, EpCAM/CD326 and NCAM/CD56. Molecular and functional assays reveal that the earliest CD326-CD56+ cells, generated from hESCs in the presence of activin A, BMP4, VEGF, and FGF2, represent a multipotent mesoderm-committed progenitor population. CD326-CD56+ progenitors are unique in their ability to generate all mesodermal lineages including hematopoietic, endothelial, mesenchymal (bone, cartilage, fat, fibroblast), smooth muscle, and cardiomyocytes, while lacking the pluripotency of hESCs. CD326-CD56+ cells are the precursors of previously reported, more lineage-restricted mesodermal progenitors. These findings present a unique approach to study how germ layer specification is regulated and offer a promising target for tissue engineering.

Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells.

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using an genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal ß- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of the already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

Distinct human skeletal muscle-derived CD90 progenitor subsets for myo-fibro-adipogenic disease modeling and treatment in multiplexed conditions.

Chronic muscle injuries, such as massive rotator cuff tears, are associated with progressive muscle wasting, fibrotic scarring, and intramuscular fat accumulation. While progenitor cell subsets are usually studied in culture conditions that drive either myogenic, fibrogenic, or adipogenic differentiation, it is still unknown how combined myo-fibro-adipogenic signals, which are expected to occur in vivo, modulate progenitor differentiation. We therefore evaluated the differentiation potential of retrospectively generated subsets of primary human muscle mesenchymal progenitors in multiplexed conditions in the presence or absence of 423F drug, a modulator of gp130 signaling. We identified a novel CD90+CD56- non-adipogenic progenitor subset that maintained a lack of adipogenic potential in single and multiplexed myo-fibro-adipogenic culture conditions. CD90-CD56- demarcated fibro-adipogenic progenitors (FAP) and CD56+CD90+ progenitors were typified as myogenic. These human muscle subsets exhibited varying degrees of intrinsically regulated differentiation in single and mixed induction cultures. Modulation of gp130 signaling via 423F drug mediated muscle progenitor differentiation in a dose-, induction-, and cell subset-dependent manner and markedly decreased fibro-adipogenesis of CD90-CD56- FAP. Conversely, 423F promoted myogenesis of CD56+CD90+ myogenic subset, indicated by increased myotube diameter and number of nuclei per myotube. 423F treatment eliminated FAP-derived mature adipocytes from mixed adipocytes-FAP cultures but did not modify the growth of non-differentiated FAP in these cultures. Collectively, these data demonstrate that capability of myogenic, fibrogenic, or adipogenic differentiation is largely dependent on the intrinsic features of cultured subsets, and that the degree of lineage differentiation varies when signals are multiplexed. Moreover, our tests performed in primary human muscle cultures reveal and confirm the potential triple-therapeutic effects of 423F drug which simultaneously attenuates degenerative fibrosis, fat accumulation and promotes myo-regeneration.

Stability analysis of tranexamic acid in the presence of various antiseptic solutions.

Tranexamic acid (TXA) effectively reduces blood loss and transfusion risk during total joint arthroplasty. Additionally, intraoperative irrigation with various antiseptic solutions is often utilized for the management and prevention of surgical site infection. However, interactions between various antiseptic solutions and TXA have not been investigated. The purpose of this in vitro study is to evaluate the stability of TXA in the presence of common orthopedic antiseptic solutions. Five antiseptic solutions-0.1% chlorhexidine (CHX) gluconate, 10% povidone-iodine (BTD), 0.5% sodium hypochlorite (Dakin's), 3% hydrogen peroxide (H2 O2 ), and 1.5% H2 O2 -and a 0.9% normal saline (NS) control were obtained. A stock 100 mg/ml TXA solution was diluted in each antiseptic solution to a concentration of 10.0 mg/ml to generate reference standard and stability samples. TXA stability in each solution was measured using high performance liquid chromatography at t = 0 and t = 120 min and reported as mean percent of theoretical concentration (MPT) with associated relative standard deviation (RSD). All experiments were performed in triplicate at room temperature. At t = 0 min, TXA remained stable when mixed with 0.9% NS, 0.1% CHX, 10% BTD, 3% H2 O2 , and 1.5% H2 O2 (MPT range: 102.0%-105.0%, RSD range: 0.80%-2.92%). Only 0.5% Dakin's led to significant degradation of TXA at t = 0 min (MPT: 14.3%, RSD:1.28%). At t = 120 min, TXA stability persisted for all compounds except Dakin's 0.5% (MPT: 18.4%, RSD: 28.7%). TXA efficacy may be significantly diminished when 0.5% Dakin's is used as an intraoperative irrigation solution. CHX, BTD, and H2 O2 do not degrade TXA.

STAT3 promotes a youthful epigenetic state in articular chondrocytes.

Epigenetic mechanisms guiding articular cartilage regeneration and age-related disease such as osteoarthritis (OA) are poorly understood. STAT3 is a critical age-patterned transcription factor highly active in fetal and OA chondrocytes, but the context-specific role of STAT3 in regulating the epigenome of cartilage cells remain elusive. In this study, DNA methylation profiling was performed across human chondrocyte ontogeny to build an epigenetic clock and establish an association between CpG methylation and human chondrocyte age. Exposure of adult chondrocytes to a small molecule STAT3 agonist decreased DNA methylation, while genetic ablation of STAT3 in fetal chondrocytes induced global hypermethylation. CUT&RUN assay and subsequent transcriptional validation revealed DNA methyltransferase 3 beta (DNMT3B) as one of the putative STAT3 targets in chondrocyte development and OA. Functional assessment of human OA chondrocytes showed the acquisition of progenitor-like immature phenotype by a significant subset of cells. Finally, conditional deletion of Stat3 in cartilage cells increased DNMT3B expression in articular chondrocytes in the knee joint in vivo and resulted in a more prominent OA progression in a post-traumatic OA (PTOA) mouse model induced by destabilization of the medial meniscus (DMM). Taken together these data reveal a novel role for STAT3 in regulating DNA methylation in cartilage development and disease. Our findings also suggest that elevated levels of active STAT3 in OA chondrocytes may indicate an intrinsic attempt of the tissue to regenerate by promoting a progenitor-like phenotype. However, it is likely that chronic activation of this pathway, induced by IL-6 cytokines, is detrimental and leads to tissue degeneration.

Inhibition of a signaling modality within the gp130 receptor enhances tissue regeneration and mitigates osteoarthritis.

Adult mammals are incapable of multitissue regeneration, and augmentation of this potential may shift current therapeutic paradigms. We found that a common co-receptor of interleukin 6 (IL-6) cytokines, glycoprotein 130 (gp130), serves as a major nexus integrating various context-specific signaling inputs to either promote regenerative outcomes or aggravate disease progression. Via genetic and pharmacological experiments in vitro and in vivo, we demonstrated that a signaling tyrosine 814 (Y814) within gp130 serves as a major cellular stress sensor. Mice with constitutively inactivated Y814 (F814) were resistant to surgically induced osteoarthritis as reflected by reduced loss of proteoglycans, reduced synovitis, and synovial fibrosis. The F814 mice also exhibited enhanced regenerative, not reparative, responses after wounding in the skin. In addition, pharmacological modulation of gp130 Y814 upstream of the SRC and MAPK circuit by a small molecule, R805, elicited a protective effect on tissues after injury. Topical administration of R805 on mouse skin wounds resulted in enhanced hair follicle neogenesis and dermal regeneration. Intra-articular administration of R805 to rats after medial meniscal tear and to canines after arthroscopic meniscal release markedly mitigated the appearance of osteoarthritis. Single-cell sequencing data demonstrated that genetic and pharmacological modulation of Y814 resulted in attenuation of inflammatory gene signature as visualized by the anti-inflammatory macrophage and nonpathological fibroblast subpopulations in the skin and joint tissue after injury. Together, our study characterized a molecular mechanism that, if manipulated, enhances the intrinsic regenerative capacity of tissues through suppression of a proinflammatory milieu and prevents pathological outcomes in injury and disease.

Dysregulated gene expression during hematopoietic differentiation from human embryonic stem cells.

The generation of hematopoietic cells from human embryonic stem cells (hESC) has raised the possibility of using hESC as an alternative donor source for transplantation. However, functional defects identified in hESC-derived cells limit their use for full lymphohematopoietic reconstitution. The purpose of the present study was to define and quantitate key functional and molecular differences between CD34(+) hematopoietic progenitor subsets derived from hESC and CD34(+) subsets from umbilical cord blood (UCB) representing definitive hematopoiesis. Two distinct sub-populations were generated following mesodermal differentiation from hESC, a CD34(bright) (hematoendothelial) and CD34(dim) (hematopoietic-restricted) subset. Limiting dilution analysis revealed profound defects in clonal proliferation relative to UCB particularly in B lymphoid conditions. Transcription factors normally expressed at specific commitment stages during B lymphoid development from UCB-CD34(+) cells were aberrantly expressed in hESC-derived CD34(+) cells. Moreover, strong negative regulators of lymphopoiesis such as the adaptor protein LNK and CCAAT/enhancer-binding protein-α (CEBPα), were exclusively expressed in hESC-CD34(+) subsets. Knockdown of LNK lead to an increase in hematopoietic progenitors generated from hESCs. The aberrant molecular profile seen in hESC-CD34(+) cells represents persistence of transcripts first expressed in undifferentiated hESC and/or CD326-CD56(+) mesoderm progenitors, and may contribute to the block in definitive hematopoiesis from hESC.

A new mouse model of post-traumatic joint injury allows to identify the contribution of Gli1+ mesenchymal progenitors in arthrofibrosis and acquired heterotopic endochondral ossification.

Complex injury and open reconstructive surgeries of the knee often lead to joint dysfunction that may alter the normal biomechanics of the joint. Two major complications that often arise are excessive deposition of fibrotic tissue and acquired heterotopic endochondral ossification. Knee arthrofibrosis is a fibrotic joint disorder where aberrant buildup of scar tissue and adhesions develop around the joint. Heterotopic ossification is ectopic bone formation around the periarticular tissues. Even though arthrofibrosis and heterotopic ossification pose an immense clinical problem, limited studies focus on their cellular and molecular mechanisms. Effective cell-targeted therapeutics are needed, but the cellular origin of both knee disorders remains elusive. Moreover, all the current animal models of knee arthrofibrosis and stiffness are developed in rats and rabbits, limiting genetic experiments that would allow us to explore the contribution of specific cellular targets to these knee pathologies. Here, we present a novel mouse model where surgically induced injury and hyperextension of the knee lead to excessive deposition of disorganized collagen in the meniscus, synovium, and joint capsule in addition to formation of extra-skeletal bone in muscle and soft tissues within the joint capsule. As a functional outcome, arthrofibrosis and acquired heterotopic endochondral ossification coupled with a significant increase in total joint stiffness were observed. By employing this injury model and genetic lineage tracing, we also demonstrate that Gli1+ mesenchymal progenitors proliferate after joint injury and contribute to the pool of fibrotic cells in the synovium and ectopic osteoblasts within the joint capsule. These findings demonstrate that Gli1+ cells are a major cellular contributor to knee arthrofibrosis and acquired heterotopic ossification that manifest after knee injury. Our data demonstrate that genetic manipulation of Gli1+ cells in mice may offer a platform for identification of novel therapeutic targets to prevent knee joint dysfunction after chronic injury.

Cross-Communication Between Knee Osteoarthritis and Fibrosis: Molecular Pathways and Key Molecules.

Knee fibrosis is characterized by the presence of excessive connective tissue due to dysregulated fibroblast activation following local or systemic tissue damage. Knee fibrosis constitutes a major clinical problem in orthopaedics due to the severe limitation in the knee range of motion that leads to compromised function and patient disability. Knee osteoarthritis is an extremely common orthopedic condition that is associated with patient disability and major costs to the health-care systems worldwide. Although knee fibrosis and osteoarthritis (OA) have traditionally been perceived as two separate pathologic entities, recent research has shown common ground between the pathophysiologic processes that lead to the development of these two conditions. The purpose of this review was to identify the pathophysiologic pathways as well as key molecules that are implicated in the development of both knee OA and knee fibrosis in order to understand the relationship between the two diagnoses and potentially identify novel therapeutic targets.

gp130/STAT3 signaling is required for homeostatic proliferation and anabolism in postnatal growth plate and articular chondrocytes.

Growth of long bones and vertebrae is maintained postnatally by a long-lasting pool of progenitor cells. Little is known about the molecular mechanisms that regulate the output and maintenance of the cells that give rise to mature cartilage. Here we demonstrate that postnatal chondrocyte-specific deletion of a transcription factor Stat3 results in severely reduced proliferation coupled with increased hypertrophy, growth plate fusion, stunting and signs of progressive dysfunction of the articular cartilage. This effect is dimorphic, with females more strongly affected than males. Chondrocyte-specific deletion of the IL-6 family cytokine receptor gp130, which activates Stat3, phenocopied Stat3-deletion; deletion of Lifr, one of many co-receptors that signals through gp130, resulted in a milder phenotype. These data define a molecular circuit that regulates chondrogenic cell maintenance and output and reveals a pivotal positive function of IL-6 family cytokines in the skeletal system with direct implications for skeletal development and regeneration.