Mesenchymal stromal cells as conductors of adipose tissue remodeling.

Adipose tissue exhibits a remarkable capacity to expand, contract, and remodel in response to changes in physiological and environmental conditions. Here, we describe recent advances in our understanding of how functionally distinct tissue-resident mesenchymal stromal cell subpopulations orchestrate several aspects of physiological and pathophysiological adipose tissue remodeling, with a particular focus on the adaptations that occur in response to changes in energy surplus and environmental temperature. The study of adipose tissue remodeling provides a vehicle to understand the functional diversity of stromal cells and offers a lens through which several generalizable aspects of tissue reorganization can be readily observed.

Lymph Node Mesenchymal and Endothelial Stromal Cells Cooperate via the RANK-RANKL Cytokine Axis to Shape the Sinusoidal Macrophage Niche.

Tissue-resident macrophages are receptive to specific signals concentrated in cellular niches that direct their cell differentiation and maintenance genetic programs. Here, we found that deficiency of the cytokine RANKL in lymphoid tissue organizers and marginal reticular stromal cells of lymph nodes resulted in the loss of the CD169+ sinusoidal macrophages (SMs) comprising the subcapsular and the medullary subtypes. Subcapsular SM differentiation was impaired in mice with targeted RANK deficiency in SMs. Temporally controlled RANK removal in lymphatic endothelial cells (LECs) revealed that lymphatic RANK activation during embryogenesis and shortly after birth was required for the differentiation of both SM subtypes. Moreover, RANK expression by LECs was necessary for SM restoration after inflammation-induced cell loss. Thus, cooperation between mesenchymal cells and LECs shapes a niche environment that supports SM differentiation and reconstitution after inflammation.

Extraocular muscle stem cells exhibit distinct cellular properties associated with non-muscle molecular signatures.

Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and single cell transcriptomic analysis to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogeninntdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells.

Matrix stiffness-dependent regulation of immunomodulatory genes in human MSCs is associated with the lncRNA CYTOR.

Cell-matrix interactions in 3D environments significantly differ from those in 2D cultures. As such, mechanisms of mechanotransduction in 2D cultures are not necessarily applicable to cell-encapsulating hydrogels that resemble features of tissue architecture. Accordingly, the characterization of molecular pathways in 3D matrices is expected to uncover insights into how cells respond to their mechanical environment in physiological contexts, and potentially also inform hydrogel-based strategies in cell therapies. In this study, a bone marrow-mimetic hydrogel was employed to systematically investigate the stiffness-responsive transcriptome of mesenchymal stromal cells. High matrix rigidity impeded integrin-collagen adhesion, resulting in changes in cell morphology characterized by a contractile network of actin proximal to the cell membrane. This resulted in a suppression of extracellular matrix-regulatory genes involved in the remodeling of collagen fibrils, as well as the upregulation of secreted immunomodulatory factors. Moreover, an investigation of long noncoding RNAs revealed that the cytoskeleton regulator RNA (CYTOR) contributes to these 3D stiffness-driven changes in gene expression. Knockdown of CYTOR using antisense oligonucleotides enhanced the expression of numerous mechanoresponsive cytokines and chemokines to levels exceeding those achievable by modulating matrix stiffness alone. Taken together, our findings further our understanding of mechanisms of mechanotransduction that are distinct from canonical mechanotransductive pathways observed in 2D cultures.

Lymphatic Endothelial Cells Control Initiation of Lymph Node Organogenesis.

Lymph nodes (LNs) are strategically situated throughout the body at junctures of the blood vascular and lymphatic systems to direct immune responses against antigens draining from peripheral tissues. The current paradigm describes LN development as a programmed process that is governed through the interaction between mesenchymal lymphoid tissue organizer (LTo) cells and hematopoietic lymphoid tissue inducer (LTi) cells. Using cell-type-specific ablation of key molecules involved in lymphoid organogenesis, we found that initiation of LN development is dependent on LTi-cell-mediated activation of lymphatic endothelial cells (LECs) and that engagement of mesenchymal stromal cells is a succeeding event. LEC activation was mediated mainly by signaling through receptor activator of NF-κB (RANK) and the non-canonical NF-κB pathway and was steered by sphingosine-1-phosphate-receptor-dependent retention of LTi cells in the LN anlage. Finally, the finding that pharmacologically enforced interaction between LTi cells and LECs promotes ectopic LN formation underscores the central LTo function of LECs.

Molecular and cellular mechanisms of chemoresistance in paediatric pre-B cell acute lymphoblastic leukaemia.

Paediatric patients with relapsed B cell acute lymphoblastic leukaemia (B-ALL) have poor prognosis, as relapse-causing clones are often refractory to common chemotherapeutics. While the molecular mechanisms leading to chemoresistance are varied, significant evidence suggests interactions between B-ALL blasts and cells within the bone marrow microenvironment modulate chemotherapy sensitivity. Importantly, bone marrow mesenchymal stem cells (BM-MSCs) and BM adipocytes are known to support B-ALL cells through multiple distinct molecular mechanisms. This review discusses the contribution of integrin-mediated B-ALL/BM-MSC signalling and asparagine supplementation in B-ALL chemoresistance. In addition, the role of adipocytes in sequestering anthracyclines and generating a BM niche favourable for B-ALL survival is explored. Furthermore, this review discusses the role of BM-MSCs and adipocytes in promoting a quiescent and chemoresistant B-ALL phenotype. Novel treatments which target these mechanisms are discussed herein, and are needed to improve dismal outcomes in patients with relapsed/refractory disease.

Human mesenchymal stromal cells ameliorate cisplatin-induced acute and chronic kidney injury via TSG-6.

Cisplatin is widely used in tumor chemotherapy, but nephrotoxicity is an unavoidable side effect of cisplatin. Several studies have demonstrated that mesenchymal stromal cells (MSCs) ameliorate cisplatin-induced kidney injury, but the underlying mechanisms are unknown. In this study, the cisplatin-induced kidney injury mouse model was established by subjecting a single intraperitoneal injection with cisplatin. One hour before cisplatin injection, the mice received human bone marrow MSCs (hBM-MSCs) with or without siRNA-transfection, recombinant human tumor necrosis factor-α-stimulated gene/protein 6 (rhTSG-6), or PBS through the tail vein. In addition, cisplatin-stimulated HK-2 cells were treated with hBM-MSCs or rhTSG-6. Human BM-MSCs treatment remarkably ameliorated cisplatin-induced acute and chronic kidney injury, as evidenced by significant reductions in serum creatinine (Scr), blood urea nitrogen, tubular injury, collagen deposition, α-smooth muscle actin accumulation, as well as inflammatory responses, and by remarkable increased anti-inflammatory factor expression and Treg cells infiltration in renal tissues. Furthermore, we found that only a few hBM-MSCs engrafted into damaged kidney and that the level of human TSG-6 in the serum of mice increased significantly following hBM-MSCs administration. Moreover, hBM-MSCs significantly increased the viability of damaged HK-2 cells and decreased the levels of inflammatory cytokines in the culture supernatant. However, the knockdown of the TSG-6 gene in hBM-MSCs significantly attenuated their beneficial effects in vivo and in vitro. On the contrary, treated with rhTSG-6 achieved similar beneficial effects of hBM-MSCs. Our results indicate that systemic administration of hBM-MSCs alleviates cisplatin-induced acute and chronic kidney injury in part by paracrine TSG-6 secretion.

Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by 2 inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.

Immunomodulatory potential of cytokine-licensed human bone marrow-derived mesenchymal stromal cells correlates with potency marker expression profile.

Cytokine(s) pre-activation/licensing is an effective way to enhance the immunomodulatory potency of mesenchymal stromal cells (MSCs). Currently, IFN-γ licensing received the most attention in comparison with other cytokines. After licensing human bone marrow-derived MSCs with pro-/anti-inflammatory cytokines IFN-γ, IL-1ß, TNF-α, TGF-ß1 alone or in combination, the in-vitro immunomodulatory potency of these MSCs was studied by incubating with allogeneic T cells and macrophage-like THP-1 cells. In addition, immunomodulation-related molecules filtered by bioinformatics, complement 1 subcomponent (C1s) and interferon-induced GTP-binding protein Mx2 (MX2), were studied to verify whether to reflect the immunomodulatory potency. Herein, we reported that different cytokines cause different effects on the function of MSC. While TGF-ß1 licensing enhances the capacity of MSCs to induce T cells with an immunosuppressive phenotype, IFN-γ-licensing strengthens the inhibitory effect of MSC on T cell proliferation. Both TGF-ß1 and IFN-γ licensing can enhance the effect of MSC on reducing the expression of pro-inflammatory cytokines by M1 macrophage-like THP-1 cells. Interestingly, IFN-γ upregulates potential potency markers extracellular C1s and kynurenine (KYN) and intracellular MX2. These three molecules have the potential to reflect mesenchymal stromal cell immunomodulatory potency. In addition, we reported that there is a synergistic effect of TGF-ß1 and IFN-γ in immunomodulation.

Mesenchymal Stromal Cell Transplantation Ameliorates Fibrosis and microRNA Dysregulation in Skeletal Muscle Ischaemia.

Peripheral arterial disease (PAD) is associated with lower-extremity muscle wasting. Hallmark features of PAD-associated skeletal muscle pathology include loss of skeletal muscle mass, reduced strength and physical performance, increased inflammation, fibrosis, and adipocyte infiltration. At the molecular level, skeletal muscle ischaemia has also been associated with gene and microRNA (miRNA) dysregulation. Mesenchymal stromal cells (MSCs) have been shown to enhance muscle regeneration and improve muscle function in various skeletal muscle injuries. This study aimed to evaluate the effects of intramuscularly delivered human umbilical cord-derived MSCs (hUC-MSCs) on skeletal muscle ischaemia. Herein, we report an hUC-MSC-mediated amelioration of ischaemia-induced skeletal muscle atrophy and function via enhancement of myofibre regeneration, reduction of tissue inflammation, adipocyte accumulation, and tissue fibrosis. These changes were observed in the absence of cell-mediated enhancement of blood flow recovery as measured by Laser Doppler imaging. Furthermore, reduced tissue fibrosis in the hUC-MSC-treated group was associated with upregulation of miR-1, miR-133a, and miR-29b and downregulation of targeted pro-fibrotic genes such as Col1a1 and Fn1. Our results support the use of hUC-MSCs as a novel approach to reduce fibrosis and promote skeletal muscle regeneration after ischaemic injury in patients with PAD.

Human Umbilical Cord Mesenchymal Stromal Cell-Derived Exosomes Alleviate Hypoxia-Induced Pulmonary Arterial Hypertension in Mice Via Macrophages.

Pulmonary hypertension (PH) is an intractable, severe, and progressive cardiopulmonary disease. Recent findings suggest that human umbilical cord mesenchymal stromal cells (HUCMSCs) and HUCMSC-derived exosomes (HUCMSC-Exos) possess potential therapeutic value for PH. However, whether they have beneficial effects on hypoxic pulmonary hypertension (HPH) is unclear. Exos are released into the extracellular environment by the fusion of intracellular multivesicular bodies with the cell membrane, and they play an important role in cellular communication. Exos ameliorate immune inflammation levels, alter macrophage phenotypes, regulate mitochondrial metabolic function, and inhibit pulmonary vascular remodeling, thereby improving PH. Macrophages are important sources of cytokines and other transmitters and can promote the release of cytokines, vasoactive molecules, and reactive oxygen species, all of which are associated with pulmonary vascular remodeling. Therefore, the aim of this study was to investigate whether HUCMSC-Exos could improve the lung inflammatory microenvironment and inhibit pulmonary vascular remodeling by targeting macrophages and identifying the underlying mechanisms. The results showed that HUCMSC-Exos promoted M2 macrophage polarization, decreased pro-inflammatory factors, increased IL-10 levels, and inhibited IL-33/ST2 axis expression, thereby inhibiting hypoxia-induced proliferation of pulmonary artery smooth muscle cells and ameliorating HPH.

Placenta-Derived Decidua Stromal Cells: A New Frontier in the Therapy of Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (GVHD) is a frequent and potentially life-threatening complication following allogeneic hematopoietic cell transplantation (HCT). Mesenchymal stromal cells (MSCs), rare precursors found in all body tissues, possess immunosuppressive properties and can inhibit alloreactivity both in vitro and in vivo. Two decades ago, we introduced bone marrow-derived (BM) MSCs as a novel therapy for acute GVHD. While some patients responded to BM-MSCs, the response was not universal. Commercially available BM-MSCs are now used for acute GVHD treatment in Canada, Japan, and New Zealand. The fetus is protected from the mother's immune system by the placenta, and our research found that placenta-derived decidua stromal cells (DSCs) offer a stronger immunosuppressive effect than other sources of stromal cells. Safety studies in rabbits, rats, mice, and humans have shown negligible or no side effects from BM-MSCs or DSCs. In a phase I/II trial for severe acute GVHD, we treated 21 patients (median age, 49 years; range 1.6-72 years) with severe biopsy-proven gastrointestinal acute GVHD. The median cell dose of DSCs was 1.2 × 106 (range 0.9-2.9) cells/kg body weight, with a median of 2 (range 1-6) infusions given 1 week apart. The cell viability of DSCs was 93% (range, 69%-100%), and the median cell passage number was 4 (range, 2-4). All patients responded, with a complete response of acute GVHD in 11 patients and partial response in 10 and 1-year survival of 81%. Randomized trials are needed to prove the superiority of DSCs compared to ruxolitinib and/or other novel immunosuppressive therapies.

Mesenchymal stromal cells protect tissues from Th1 immune responses via IL-11 secretion.

Mesenchymal stromal cells (MSCs) have been shown to modulate the function of various subsets of T cells such as naïve CD4+ T cells and IFNγ+CD4+ Th1 cells; however, mechanisms underlying this regulation have not been fully deciphered. Our in vitro culture assays demonstrate that MSCs suppress the activation and function of CD4+ T cells by secreting interleukin 11, and neutralization of IL11 abrogates MSC-mediated suppression of CD4+ T cell function. Moreover, delayed-type, exogenous supplementation of IL11 significantly suppressed IFNγ+ expression by Th1 cells. Th1 and CD8+ cells play central roles in T cell-mediated tissue damage. Using a murine model of hypersensitivity response to study T cell-mediated tissue damage, we show that silencing IL11 in MSCs significantly abates the capacity of MSCs to suppress the generation of IFNγ-secreting CD4+ and CD8+ cells, failing to prevent T cell-mediated tissue inflammation and tissue damage.

Carbon monoxide-induced autophagy enhances human mesenchymal stromal cell function via paracrine actions in murine polymicrobial sepsis.

Sepsis is a life-threatening process due to organ dysfunction resulting from severe infections. Mesenchymal stromal cells (MSCs) are being investigated as therapy for sepsis, along with conditioning regimens to improve their function. Carbon monoxide (CO) gas, which is cytoprotective at low doses, induces autophagy and is a mediator of inflammation. We evaluated CO-induced autophagy in human MSCs (hMSCs), and its impact on cell function in murine cecal ligation and puncture. Conditioning of hMSCs with CO ex vivo resulted in enhanced survival and bacterial clearance in vivo, and neutrophil phagocytosis of bacteria in vitro. Decreased neutrophil infiltration and less parenchymal cell death in organs were associated with increased macrophage efferocytosis of apoptotic neutrophils, promoting resolution of inflammation. These CO effects were lost when the cells were exposed to autophagy inhibition prior to gas exposure. When assessing paracrine actions of CO-induced autophagy, extracellular vesicles (EVs) were predominantly responsible. CO had no effect on EV production, but altered their miRNA cargo. Increased expression of miR-145-3p and miR-193a-3p by CO was blunted with disruption of autophagy, and inhibitors of these miRNAs led to a loss of neutrophil phagocytosis and macrophage efferocytosis. Collectively, CO-induced autophagy enhanced hMSC function during sepsis via paracrine actions of MSC-derived EVs.

CRISPR-mediated Sox9 activation and RelA inhibition enhance cell therapy for osteoarthritis.

Osteoarthritis (OA) is a painful and debilitating disease affecting over 500 million people worldwide. Intraarticular injection of mesenchymal stromal cells (MSCs) shows promise for the clinical treatment of OA, but the lack of consistency in MSC preparation and application makes it difficult to further optimize MSC therapy and to properly evaluate the clinical outcomes. In this study, we used Sox9 activation and RelA inhibition, both mediated by the CRISPR-dCas9 technology simultaneously, to engineer MSCs with enhanced chondrogenic potential and downregulated inflammatory responses. We found that both Sox9 and RelA could be fine-tuned to the desired levels, which enhances the chondrogenic and immunomodulatory potentials of the cells. Intraarticular injection of modified cells significantly attenuated cartilage degradation and palliated OA pain compared with the injection of cell culture medium or unmodified cells. Mechanistically, the modified cells promoted the expression of factors beneficial to cartilage integrity, inhibited the production of catabolic enzymes in osteoarthritic joints, and suppressed immune cells. Interestingly, a substantial number of modified cells could survive in the cartilaginous tissues including articular cartilage and meniscus. Together, our results suggest that CRISPR-dCas9-based gene regulation is useful for optimizing MSC therapy for OA.

Cell-based Therapies for Acute Respiratory Distress Syndrome: Where Are We Now?

There is considerable interest in the potential for cell-based therapies, particularly mesenchymal stromal cells (MSCs) and their products, as a therapy for acute respiratory distress syndrome (ARDS). MSCs exert effects via diverse mechanisms including reducing excessive inflammation by modulating neutrophil, macrophage and T-cell function, decreasing pulmonary permeability and lung edema, and promoting tissue repair. Clinical studies indicate that MSCs are safe and well tolerated, with promising therapeutic benefits in specific clinical settings, leading to regulatory approvals of MSCs for specific indications in some countries.This perspective reassesses the therapeutic potential of MSC-based therapies for ARDS given insights from recent cell therapy trials in both COVID-19 and in 'classic' ARDS, and discusses studies in graft-vs.-host disease, one of the few licensed indications for MSC therapies. We identify important unknowns in the current literature, address challenges to clinical translation, and propose an approach to facilitate assessment of the therapeutic promise of MSC-based therapies for ARDS.

Early bone marrow alterations in patients with adenosine deaminase 2 deficiency across disease phenotypes and severities.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is a complex monogenic disease caused by recessive mutations in the ADA2 gene. DADA2 exhibits a broad clinical spectrum encompassing vasculitis, immunodeficiency, and hematologic abnormalities. Yet, the impact of DADA2 on the bone marrow (BM) microenvironment is largely unexplored. OBJECTIVE: This study comprehensively examined the BM and peripheral blood of pediatric and adult patients with DADA2 presenting with rheumatologic/immunologic symptoms or severe hematologic manifestations. METHODS: Immunophenotyping of hematopoietic stem cells (HSCs), progenitor cells, and mature cell populations was performed for 18 patients with DADA2. We also conducted a characterization of mesenchymal stromal cells. RESULTS: Our study revealed a significant decrease in primitive HSCs and progenitor cells, alongside their reduced clonogenic capacity and multilineage differentiation potential. These BM defects were evident in patients with both severe and nonsevere hematologic manifestations, including pediatric patients, demonstrating that BM disruption can emerge silently and early on, even in patients who do not show obvious hematologic symptoms. Beyond stem cells, there was a reduction in mature cell populations in the BM and peripheral blood, affecting myeloid, erythroid, and lymphoid populations. Furthermore, BM mesenchymal stromal cells in patients with DADA2 exhibited reduced clonogenic and proliferation capabilities and were more prone to undergo cellular senescence marked by elevated DNA damage. CONCLUSIONS: Our exploration into the BM landscape of patients with DADA2 sheds light on the critical hematologic dimension of the disease and emphasizes the importance of vigilant monitoring, even in the case of subclinical presentation.

Perivascular stromal cells: Directors of tissue immune niches.

Perivascular niches are specialized microenvironments where stromal and immune cells interact with vasculature to monitor tissue status. Adventitial perivascular niches surround larger blood vessels and other boundary sites, supporting collections of immune cells, stromal cells, lymphatics, and neurons. Adventitial fibroblasts (AFs), a subtype of mesenchymal stromal cell, are the dominant constituents in adventitial spaces, regulating vascular integrity while organizing the accumulation and activation of a variety of interacting immune cells. In contrast, pericytes are stromal mural cells that support microvascular capillaries and surround organ-specific parenchymal cells. Here, we outline the unique immune and non-immune composition of perivascular tissue immune niches, with an emphasis on the heterogeneity and immunoregulatory functions of AFs and pericytes across diverse organs. We will discuss how perivascular stromal cells contribute to the regulation of innate and adaptive immune responses and integrate immunological signals to impact tissue health and disease.

Four sides to the story: A proteomic comparison of liquid-phase and matrix-bound extracellular vesicles in 2D and 3D cell cultures.

Multipotent mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) play important roles in cellular communication and are extensively studied as promising therapeutic agents. While there is a substantial pool of studies on liquid-phase EVs, data on EVs bound to the extracellular matrix (ECM) is lacking. There is also an emerging trend of accumulating and comparing data on characteristics of EVs obtained in different culturing conditions. Aiming to reveal proteomic signatures of EVs obtained from conditioned media and ECM of MSCs cultured in 2D and 3D conditions, we performed liquid chromatography with tandem mass spectrometry. Bioinformatic analysis revealed common patterns in proteomic composition of liquid-phase EVs and matrix-bound vesicles (MBVs), namely extracellular environment organization, immune, and transport pathways enrichment. However, extracellular environmental organization pathways are more enriched in liquid-phase EVs than in MBVs, while MBVs proteins noticeably enrich enzymatic pathways. Furthermore, each type of EVs from 2D and 3D cultures has a unique differential abundance profile. We have also performed comparative functional assays, namely scratch assay to assess EVs effect on cell migration and tubulogenesis assay to evaluate EVs angiogenic potential. We found that both liquid-phase EVs and MBVs enhance cell migration, while angiogenic potential is higher in MBVs. Results of the present study suggest that while both liquid-phase EVs and MBVs have therapeutic potential, some unique features of each subgroup may determine optimal areas of their application.

Mechanoinduction of PTHrP/cAMP-signaling governs proteoglycan production in mesenchymal stromal cell-derived neocartilage.

Abnormal mechanical loading is one of the major risk factors for articular cartilage degeneration. Engineered mesenchymal stromal cell (MSC)-derived cartilage holds great promise for cell-based cartilage repair. However, physiological loading protocols were shown to reduce matrix synthesis of MSC-derived neocartilage in vitro and the regulators of this undesired mechanoresponse remain poorly understood. Parathyroid hormone-related protein (PTHrP) is involved in cartilage development and can affect extracellular matrix (ECM) production during MSC chondrogenesis opposingly, depending on a continuous or transient exposure. PTHrP is induced by various mechanical cues in multiple tissues and species; but whether PTHrP is regulated in response to loading of human engineered neocartilage and may affect matrix synthesis in a positive or negative manner is unknown. The aim of this study was to investigate whether dynamic loading adjusts PTHrP-signaling in human MSC-derived neocartilage and whether it regulates matrix synthesis and other factors involved in the MSC mechanoresponse. Interestingly, MSC-derived chondrocytes significantly upregulated PTHrP mRNA (PTHLH) expression along with its second messenger cAMP in response to loading in our custom-built bioreactor. Exogenous PTHrP(1-34) induced the expression of known mechanoresponse genes (FOS, FOSB, BMP6) and significantly decreased glycosaminoglycan (GAG) and collagen synthesis similar to loading. The adenylate-cyclase inhibitor MDL-12,330A rescued the load-mediated decrease in GAG synthesis, indicating a direct involvement of cAMP-signaling in the reduction of ECM production. According to COL2A1-corrected hypertrophy-associated marker expression, load and PTHrP treatment shared the ability to reduce expression of MEF2C and PTH1R. In conclusion, the data demonstrate a significant mechanoinduction of PTHLH and a negative contribution of the PTHrP-cAMP signaling axis to GAG synthesis in MSC-derived chondrocytes after loading. To improve ECM synthesis and the mechanocompetence of load-exposed neocartilage, inhibition of PTHrP activity should be considered for MSC-based cartilage regeneration strategies.