Acute respiratory distress syndrome (ARDS) in COVID‐19 is associated with high mortality. Mesenchymal stem cells are known to exert immunomodulatory and anti‐inflammatory effects and could yield beneficial effects in COVID‐19 ARDS. The objective of this study was to determine safety and explore efficacy of umbilical cord mesenchymal stem cell (UC‐MSC) infusions in subjects with COVID‐19 ARDS. A double‐blind, phase 1/2a, randomized, controlled trial was performed. Randomization and stratification by ARDS severity was used to foster balance among groups. All subjects were analyzed under intention to treat design. Twenty‐four subjects were randomized 1:1 to either UC‐MSC treatment (n = 12) or the control group (n = 12). Subjects in the UC‐MSC treatment group received two intravenous infusions (at day 0 and 3) of 100 ± 20 × 106 UC‐MSCs; controls received two infusions of vehicle solution. Both groups received best standard of care. Primary endpoint was safety (adverse events [AEs]) within 6 hours; cardiac arrest or death within 24 hours postinfusion). Secondary endpoints included patient survival at 31 days after the first infusion and time to recovery. No difference was observed between groups in infusion‐associated AEs. No serious adverse events (SAEs) were observed related to UC‐MSC infusions. UC‐MSC infusions in COVID‐19 ARDS were found to be safe. Inflammatory cytokines were significantly decreased in UC‐MSC‐treated subjects at day 6. Treatment was associated with significantly improved patient survival (91% vs 42%, P = .015), SAE‐free survival (P = .008), and time to recovery (P = .03). UC‐MSC infusions are safe and could be beneficial in treating subjects with COVID‐19 ARDS.

Bone fractures and segmental bone defects are a significant source of patient morbidity and place a staggering economic burden on the healthcare system. The annual cost of treating bone defects in the US has been estimated to be $5 billion, while enormous costs are spent on bone grafts for bone injuries, tumors, and other pathologies associated with defective fracture healing. Autologous bone grafts represent the gold standard for the treatment of bone defects. However, they are associated with variable clinical outcomes, postsurgical morbidity, especially at the donor site, and increased surgical costs. In an effort to circumvent these limitations, tissue engineering and cell-based therapies have been proposed as alternatives to induce and promote bone repair. This review focuses on the recent advances in bone tissue engineering (BTE), specifically looking at its role in treating delayed fracture healing (non-unions) and the resulting segmental bone defects. Herein we discuss: (1) the processes of endochondral and intramembranous bone formation; (2) the role of stem cells, looking specifically at mesenchymal (MSC), embryonic (ESC), and induced pluripotent (iPSC) stem cells as viable building blocks to engineer bone implants; (3) the biomaterials used to direct tissue growth, with a focus on ceramic, biodegradable polymers, and composite materials; (4) the growth factors and molecular signals used to induce differentiation of stem cells into the osteoblastic lineage, which ultimately leads to active bone formation; and (5) the mechanical stimulation protocols used to maintain the integrity of the bone repair and their role in successful cell engraftment. Finally, a couple clinical scenarios are presented (non-unions and avascular necrosis—AVN), to illustrate how novel cell-based therapy approaches can be used. A thorough understanding of tissue engineering and cell-based therapies may allow for better incorporation of these potential therapeutic approaches in bone defects allowing for proper bone repair and regeneration.

ObjectiveControversy surrounds the identity and functionality of rare bone marrow–derived multipotential stromal cells (BM‐MSCs), including their differentiation capabilities, their relationship to pericytes and hematopoiesis‐supporting stromal cells, and the relevance of their culture‐expanded progeny in studies of skeletal biology and development of cell‐based therapies. The aim of this study was to clarify the nature of candidate BM‐MSCs by profiling transcripts that reflect different aspects of their putative functions in vivo.MethodsRare, sorted BM‐derived CD45−/low CD271bright (CD271) cells were analyzed using 96‐gene expression arrays focused on transcripts relevant to mesenchymal‐lineage differentiation (toward bone, cartilage, fat, or muscle), hematopoietic and stromal support, and molecules critical to skeletal homeostasis. These cells were compared to matched CD45+ CD271− hematopoietic‐lineage cells, culture‐expanded MSCs, and skin fibroblasts. When feasible, transcription was validated using flow cytometry.ResultsCD271 cells had a transcriptional profile consistent with the multiple fates of in vivo MSCs, evident from the observed simultaneous expression of osteogenic, adipogenic, pericytic, and hematopoiesis‐supporting genes (e.g., SP7 [osterix], FABP4 [fatty acid binding protein 4], ANGPT1 [angiopoietin 1], and CXCL12 [stromal cell–derived factor 1], respectively). Compared to culture‐expanded MSCs and fibroblasts, CD271 cells exhibited greater transcriptional activity, particularly with respect to Wnt‐related genes (>1,000‐fold increased expression of FRZB [secreted frizzled‐related protein 3] and WIF1 [Wnt inhibitory factor 1]). A number of transcripts were identified as novel markers of MSCs.ConclusionThe native, BM‐derived in vivo MSC population is endowed with a gene signature that is compatible with multiple functions, reflecting the topographic bone niche of these cells, and their signature is significantly different from that of culture‐expanded MSCs. This indicates that studies of the biologic functions of MSCs in musculoskeletal diseases, including osteoporosis and osteoarthritis, should focus on in vivo MSCs, rather than their culture‐adapted progeny.

Objective. To test the hypothesis that CD45 low CD271؉ bone marrow multipotential stromal cells (MSCs) are abundant in the trabecular bone niche and to explore their functional "fitness" in health and osteoarthritis (OA).Methods. Following enzymatic extraction, MSC release was evaluated using colony-forming unitfibroblast (CFU-F) and colony-forming unit-osteoblast assays, flow cytometry, and confocal microscopy. CD45 low CD271؉ cells isolated by fluorescence-activated cell sorting were enumerated and expanded under standard and clonal conditions. Their proliferative and osteogenic potencies were assessed in relation to donor age and compared with those of aspirated CD45 low CD271؉ cells. In vitro and in vivo MSC "aging" was measured using quantitative polymerase chain reaction-based telomere length analysis, and standard differentiation assays were utilized to demonstrate multipotentiality.Results. Conclusion. Our findings show that CD45 low CD271؉ MSCs are abundant in the trabecular bone cavity and indistinguishable from aspirated CD45 low CD271؉ MSCs. In OA they display agingrelated loss of proliferation but no gross osteogenic abnormality. These findings offer new opportunities for direct study of MSCs in musculoskeletal diseases without the requirement for culture expansion. They are also relevant for direct therapeutic exploitation of prospectively isolated, minimally cultured MSCs in trauma and OA.

The infrapatellar fat pad (IFP) serves as a reservoir of Mesenchymal Stem Cells (MSC), and with adjacent synovium plays key roles in joint disease including the production of Substance P (SP) affecting local inflammatory responses and transmitting nociceptive signals. Here, we interrogate human IFP-derived MSC (IFP-MSC) reaction to inflammatory and pro-fibrotic environments (cell priming by TNFα/IFNγ and TNFα/IFNγ/CTGF exposure respectively), compared with bone marrow-derived MSC (BM-MSC). Naïve IFP-MSC exhibit increased clonogenicity and chondrogenic potential compared with BM-MSC. Primed cells experienced dramatic phenotypic changes, including a sharp increase in CD10, upregulation of key immunomodulatory transcripts, and secreted growth factors/cytokines affecting key pathways (IL-10, TNF-α, MAPK, Ras and PI3K-Akt). Naïve, and more so primed MSC (both) induced SP degradation in vitro , reproduced with their supernatants and abrogated with thiorphan, a CD10 inhibitor. These findings were reproduced in vivo in a rat model of acute synovitis, where transiently engrafted human IFP-MSC induced local SP reduction. Functionally, primed IFP-MSC demonstrated sustained antagonism of activated human peripheral blood mononuclear cells (PBMC) proliferation, significantly outperforming a declining dose-dependent effect with naïve cohorts. Collectively, our in vitro and in vivo data supports cell priming as a way to enhance the immunoregulatory properties of IFP-MSC, which selectively engraft in areas of active synovitis/IFP fibrosis inducing SP degradation, resulting in a cell-based product alternative to BM-MSC to potentially treat degenerative/inflammatory joint diseases.

To date, the therapeutic efficacy of human mesenchymal stem cells (hMSCs) has been investigated in various clinical trials with moderate or, in some cases, inconsistent results. The still elusive reproducibility relates, in part, with constitutive differences in the cell preparation, translated into variable "cell potencies." Other factors include poor cell homing and survival, and age-/disease-associated host tissue impairment. It is well accepted that within in vivo niches, MSCs exist as heterogeneous cell populations with different stemness propensities and supportive functions. Phenotype-based MSC purification of homogeneous subsets can result in cell populations with distinct biological functions. In addition, preclinical studies have shown that MSC functionalization in vitro, through cell priming, can boost their immunomodulatory, trophic, and reparative capacities in vivo. Therefore, in this review, we discuss how phenotype-based MSC purification and MSC priming technologies can contribute to an improved MSC-based product for safer and more effective therapeutic applications.

Background: osteoarthritic human articular cartilage (AC)-derived cartilage cells (CCs) with same-donor bone marrow (BMSCs) and adipose tissue (ASCs)-derived mesenchymal stem cells were compared, in terms of stemness features, and secretory and immunomodulatory responses to inflammation. Methods: proteoglycan 4 (PRG4) presence was evaluated in AC and CCs. MSCs and CCs (n = 8) were cultured (P1 to P4) and characterized for clonogenicity, nanog homeobox (NANOG), and POU class 5 homeobox 1 (POU5F1) expression, immunotypification, and tri-lineage differentiation. Their basal and interleukin-1β (IL-1β)-stimulated expression of matrix metalloproteases (MMPs), tissue inhibitors (TIMPs), release of growth factors, and cytokines were analyzed, along with the immunomodulatory ability of CCs. Results: PRG4 was mainly expressed in the intact AC surface, whereas shifted to the intermediate zone in damaged cartilage and increased its expression in CCs upon culture. All cells exhibited a similar phenotype and stemness maintenance over passages. CCs showed highest chondrogenic ability, no adipogenic potential, a superior basal secretion of growth factors and cytokines, the latter further increased after inflammatory stimulation, and an immunomodulatory behavior. All stimulated cells shared an increased MMP expression without a corresponding TIMP production. Conclusion: based on the observed features, CCs obtained from pathological joints may constitute a potential tissue-specific therapeutic target or agent to improve damaged cartilage healing, especially damage caused by inflammatory/immune mediated conditions.

Background aimsMesenchymal stromal cells (MSCs) are regenerative and immuno-privileged cells that are used for both tissue regeneration and treatment of severe inflammation-related disease. For quality control of manufactured MSC batches in regard to mature fat cell contamination, a quantitative method for measuring adipogenesis is needed.MethodsFour previously proposed methods were validated with the use of bone marrow (BM) MSCs during a 21-day in vitro assay. Oil red staining was scored semiquantitatively; peroxisome proliferator activated receptor-γ and fatty acid binding protein (FABP)4 transcripts were measured by quantitative real-time polymerase chain reaction; FABP4 protein accumulation was evaluated by flow cytometry; and Nile red/4′,6-diamidino-2-phenylindole (DAPI) ratios were measured in fluorescent microplate assay. Skin fibroblasts and MSCs from fat pad, cartilage and umbilical cord were used as controls.ResultsOil red staining indicated considerable heterogeneity between BM donors and individual cells within the same culture. FABP4 transcript levels increased 100- to 5000-fold by day 21, with large donor variability observed. Flow cytometry revealed increasing intra-culture heterogeneity over time; more granular cells accumulated more FABP4 protein and Nile red fluorescence compared with less granular cells. Nile red increase in day-21 MSCs was ∼5- and 4-fold, measured by flow cytometry or microplate assay, respectively. MSC proliferation/apoptosis was accounted through the use of Nile red/DAPI ratios; adipogenesis levels in day-21 BM MSCs increased ∼13-fold, with significant correlations with oil red scoring observed for MSC from other sources.ConclusionsFlow cytometry permits the study of MSC differentiation at the single-cell level and sorting more and less mature cells from mixed cell populations. The microplate assay with the use of the Nile red/DAPI ratio provides rapid quantitative measurements and could be used as a low-cost, high-throughput method to quality-control MSC batches from different tissue sources.