Kindlin-3 mutation in mesenchymal stem cells results in enhanced chondrogenesis.

Identifying patient mutations driving skeletal development disorders has driven our understanding of bone development. Integrin adhesion deficiency disease is caused by a Kindlin-3 (fermitin family member 3) mutation, and its inactivation results in bleeding disorders and osteopenia. In this study, we uncover a role for Kindlin-3 in the differentiation of bone marrow mesenchymal stem cells (BMSCs) down the chondrogenic lineage. Kindlin-3 expression increased with chondrogenic differentiation, similar to RUNX2. BMSCs isolated from a Kindlin-3 deficient patient expressed chondrocyte markers, including SOX9, under basal conditions, which were further enhanced with chondrogenic differentiation. Rescue of integrin activation by a constitutively activated ß3 integrin construct increased adhesion to multiple extracellular matrices and reduced SOX9 expression to basal levels. Growth plates from mice expressing a mutated Kindlin-3 with the integrin binding site ablated demonstrated alterations in chondrocyte maturation similar to that seen with the human Kindlin-3 deficient BMSCs. These findings suggest that Kindlin-3 expression mirrors RUNX2 during chondrogenesis.

Intrarenal injection of mesenchymal stem cell for treatment of lupus nephritis in mice - a pilot study.

The current project is to explore feasibility of direct intra-renal injection of human bone marrow derived mesenchymal stem cells (hMSC) for treatment of lupus nephritis in mice. The treatment protocol involved aged male BXSB (20 weeks) injected with 1 × 106 hMSC unilaterally under the renal capsule. Mice were harvested after 10 weeks follow-up for postmortem exam. Controls included untreated age matched male BXSB and healthy C57Bl/6. At the end of follow-up period, the survival of treated BXSB was 10 folds higher at 62.5% compared to survival of untreated control at 6.3%. The survival of C57Bl/6 remained at 100% with or without similar treatment. The renal pathology review was most significant for decreased tissue inflammation in treated BXSB compared to untreated controls. Renal tissue expression of IL-1b, IL17 were decreased and CTLA-4 was increased by RT PCR among treated compared to untreated BXSB. Thus, direct delivery of hMSC by intrarenal injection is a promising route for treatment of lupus nephritis as shown in this xenogeneic model. Further studies -using expanded numbers of mice to include other lupus strains- are warranted to investigate the mechanisms involved and to optimize treatment protocol for safety and efficacy.

An Integrated Multi-Function Heterogeneous Biochemical Circuit for High-Resolution Electrochemistry-Based Genetic Analysis.

Modular construction of an autonomous and programmable multi-functional heterogeneous biochemical circuit that can identify, transform, translate, and amplify biological signals into physicochemical signals based on logic design principles can be a powerful means for the development of a variety of biotechnologies. To explore the conceptual validity, we design a CRISPR-array-mediated primer-exchange-reaction-based biochemical circuit cascade, which probes a specific biomolecular input, transform the input into a structurally accessible form for circuit wiring, translate the input information into an arbitrary sequence, and finally amplify the prescribed sequence through autonomous formation of a signaling concatemer. This upstream biochemical circuit is further wired with a downstream electrochemical interface, delivering an integrated bioanalytical platform. We program this platform to directly analyze the genome of SARS-CoV-2 in human cell lysate, demonstrating the capability and the utility of this unique integrated system.

Exploring the Trans-Cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor.

An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR type V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the in vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor ß1 (TGF-ß1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.

MSCs: The Sentinel and Safe-Guards of Injury.

Mesenchymal stem cells (MSCs) were originally named because they could differentiate in a variety of mesenchymal phenotypes in culture. Evidence indicates that MSCs arise from perivascular cells, pericytes, when the blood vessels are broken or inflamed. These pericyte/MSCs are situated on every blood vessel in the body. The MSCs sense the micro-environment of the injury site and secrete site-specific factors that serve several important reparative functions: first, a curtain of molecules from the front of the MSCs provide a barrier from the interrogation of the over-aggressive immune system. Second, from the back of the MSCs, a different set of bioactive agents inhibit scar formation and establish a regenerative micro-environment. Third, if bacteria are sensed by the MSCs, they produce powerful protein antibiotics that kill the bacteria on contact. Last, the MSCs surround and encyst intruding solid objects like a piece of wood (a "splinter") or other foreign objects. The MSCs act as a combination paramedic and emergency room (ER) staff to survey the damage, isolate foreign components, stabilize the injured tissues, provide antibiotics and encysting protection before a slower, medicinal sequence can be initiated to regenerate the damaged tissue. The MSCs, thus, act as sentinels to safeguard the individual from intrusion and chronic injury. A societal treatment system has evolved, paramedics and ER procedures, which mirror in a macro-sense what MSCs orchestrate in a micro-sense. Key to this new understanding is that MSCs are not "stem cells," but rather as Medicinal Signaling Cells as the therapeutic agents.

Mesenchymal stem cells regulate melanoma cancer cells extravasation to bone and liver at their perivascular niche.

Skeleton and liver are preferred organs for cancer dissemination in metastatic melanoma negatively impacting quality of life, therapeutic success and overall survival rates. At the target organ, the local microenvironment and cell-to-cell interactions between invading and resident stromal cells constitute critical components during the establishment and progression of metastasis. Mesenchymal stem cells (MSCs) possess, in addition to their cell progenitor function, a secretory capacity based on cooperativity with other cell types in injury sites including primary tumors (PT). However, their role at the target organ microenvironment during cancer dissemination is not known. We report that local MSCs, acting as pericytes, regulate the extravasation of melanoma cancer cells (MCC) specifically to murine bone marrow (BM) and liver. Intra-arterially injected wild-type MCC fail to invade those selective organs in a genetic model of perturbed pericyte coverage of the vasculature (PDGF-B(ret/ret)), similar to CD146-deficient MCC injected into wild type mice. Invading MCC interact with resident MSCs/pericytes at the perivascular space through co-expressed CD146 and Sdf-1/CXCL12-CXCR4 signaling. Implanted engineered bone structures with MSCs/pericytes deficient of either Sdf-1/CXCL12 or CD146 become resistant to invasion by circulating MCC. Collectively, the presence of MSCs/pericytes surrounding the target organ vasculature is required for efficient melanoma metastasis to BM and liver.

Platelet-Derived Growth Factor BB Enhances Osteogenesis of Adipose-Derived But Not Bone Marrow-Derived Mesenchymal Stromal/Stem Cells.

Tissue engineering using mesenchymal stem cells (MSCs) holds great promise for regenerating critically sized bone defects. While the bone marrow-derived MSC is the most widely studied stromal/stem cell type for this application, its rarity within bone marrow and painful isolation procedure have motivated investigation of alternative cell sources. Adipose-derived stromal/stem cells (ASCs) are more abundant and more easily procured; furthermore, they also possess robust osteogenic potency. While these two cell types are widely considered very similar, there is a growing appreciation of possible innate differences in their biology and response to growth factors. In particular, reports indicate that their osteogenic response to platelet-derived growth factor BB (PDGF-BB) is markedly different: MSCs responded negatively or not at all to PDGF-BB while ASCs exhibited enhanced mineralization in response to physiological concentrations of PDGF-BB. In this study, we directly tested whether a fundamental difference existed between the osteogenic responses of MSCs and ASCs to PDGF-BB. MSCs and ASCs cultured under identical osteogenic conditions responded disparately to 20 ng/ml of PDGF-BB: MSCs exhibited no difference in mineralization while ASCs produced more calcium per cell. siRNA-mediated knockdown of PDGFRß within ASCs abolished their ability to respond to PDGF-BB. Gene expression was also different; MSCs generally downregulated and ASCs generally upregulated osteogenic genes in response to PDGF-BB. ASCs transduced to produce PDGF-BB resulted in more regenerated bone within a critically sized murine calvarial defect compared to control ASCs, indicating PDGF-BB used specifically in conjunction with ASCs might enhance tissue engineering approaches for bone regeneration.

Serial transplantation and long-term engraftment of intra-arterially delivered clonally derived mesenchymal stem cells to injured bone marrow.

It has been hypothesized that mesenchymal stem cells (MSCs) home to sites of injury. Nevertheless, efficient delivery of MSCs to target organs and description of their ultimate fate remain major challenges. We provide evidence that intra-arterially (IA) injected MSCs selectively engraft from the circulation as perivascular cells in the bone marrow (BM) after a localized radiation injury. Luciferase-expressing MSCs, derived from a conditionally immortalized clone (BMC-9) representing a pure population of cells, were arterially delivered into mice irradiated in one leg. Cell distribution was measured by bioluminescent imaging and final destination assessed by luciferase immunolocalization. IA injections resulted in engraftment only in the irradiated leg where cells localize and proliferate abluminal to the BM vasculature, a phenomenon not replicated with intravenous injections or with IA injections of kidney cells harvested from the same donor used for MSCs. Furthermore, MSCs harvested from the engrafted marrow and serially transplanted retain the ability to selectively engraft at sites of injury. This study demonstrates that MSCs can serially engraft at sites of injury from the circulation, that they reside in the perivascular space, and that arterial delivery is more efficient than venous delivery for cell engraftment.

The role of CXCL12 and CCL7 chemokines in immune regulation, embryonic development, and tissue regeneration.

Chemotactic factors direct the migration of immune cells, multipotent stem cells, and progenitor cells under physiologic and pathologic conditions. Chemokine ligand 12 and chemokine ligand 7 have been identified and investigated in multiple studies for their role in cellular trafficking in the setting of tissue regeneration. Recent early phase clinical trials have suggested that these molecules may lead to clinical benefit in patients with chronic disease. Importantly, these two proteins may play additional significant roles in directing the migration of multipotent cells, such as mesenchymal stem cells and hematopoietic progenitor cells. This article reviews the functions of these two chemokines, focusing on recruitment to sites of injury, immune function modulation, and contributions to embryonic development. Additional research would provide valuable insight into the potential clinical application of these two proteins in stem cell therapy.

Growth, differentiation capacity, and function of mesenchymal stem cells expanded in serum-free medium developed via combinatorial screening.

The presence of serum in cell culture medium presents an obstacle to safe and efficient production of hMSCs for therapeutic purposes. Availability of defined medium will be crucial to elucidating the mechanism of action of hMSCs in many indications as well as a prerequisite to consistently produce cells with predictable performance characteristics. Using a bioinformatics driven approach, which we call the BD Discovery Platform, we have developed a novel serum-free medium that supports highly efficient growth while maintaining the surface markers and functional characteristics defining hMSCs. In a comparison with serum-containing and other commercially available serum-free formulations, all conditions led to expansion of cells that meet the minimal criteria for hMSCs as set by the International Society for Cellular Therapy (ISCT). However, differences in growth characteristics and gene expression patterns suggest that expansion in serum-free growth conditions can provide greater yields in a shorter time. The mRNA expression profile observed in cells grown without serum suggests upregulation of several genes implicated in hMSC function as well as downregulation of the proinflammatory cytokine IL6.

Amino Acid Uptake Limitations during Human Mesenchymal Stem Cell-Based Chondrogenesis.

A mino acids are the essential building blocks for collagen and proteoglycan, which are the main constituents for cartilage extracellular matrix (ECM). Synthesis of ECM proteins requires the uptake of various essential/nonessential amino acids. Analyzing amino acid metabolism during chondrogenesis can help to relate tissue quality to amino acid metabolism under different conditions. In our study, we studied amino acid uptake/secretion using human mesenchymal stem cell (hMSC)-based aggregate chondrogenesis in a serum-free induction medium with a defined chemical formulation. The initial glucose level and medium-change frequency were varied. Our results showed that essential amino acid uptake increased with time during hMSCs chondrogenesis for all initial glucose levels and medium-change frequencies. Essential amino acid uptake rates were initial glucose-level independent. The DNA-normalized glycosaminoglycans and hydroxyproline content of chondrogenic aggregates correlated with cumulative uptake of leucine, valine, and tryptophan regardless of initial glucose levels and medium-change frequencies. Collectively, our results show that amino acid uptake rates during in vitro chondrogenesis were insufficient to produce a tissue with an ECM content similar to that of human neonatal cartilage or adult cartilage. Furthermore, this deficiency was likely related to the downregulation of some key amino acid transporters in the cells. Such deficiency could be partially improved by increasing the amino acid availability in the chondrogenic medium by changing culture conditions.

MSCs mediate long-term efficacy in a Crohn's disease model by sustained anti-inflammatory macrophage programming via efferocytosis.

Mesenchymal stem cells (MSCs) are novel therapeutics for the treatment of Crohn's disease. However, their mechanism of action is unclear, especially in disease-relevant chronic models of inflammation. Thus, we used SAMP-1/YitFc (SAMP), a chronic and spontaneous murine model of small intestinal inflammation, to study the therapeutic effects and mechanism of action of human bone marrow-derived MSCs (hMSC). hMSC dose-dependently inhibited naïve T lymphocyte proliferation via prostaglandin E2 (PGE2) secretion and reprogrammed macrophages to an anti-inflammatory phenotype. We found that the hMSCs promoted mucosal healing and immunologic response early after administration in SAMP when live hMSCs are present (until day 9) and resulted in a complete response characterized by mucosal, histological, immunologic, and radiological healing by day 28 when no live hMSCs are present. hMSCs mediate their effect via modulation of T cells and macrophages in the mesentery and mesenteric lymph nodes (mLN). Sc-RNAseq confirmed the anti-inflammatory phenotype of macrophages and identified macrophage efferocytosis of apoptotic hMSCs as a mechanism that explains their long-term efficacy. Taken together, our findings show that hMSCs result in healing and tissue regeneration in a chronic model of small intestinal inflammation and despite being short-lived, exert long-term effects via sustained anti-inflammatory programming of macrophages via efferocytosis.

Multidisciplinary Management Strategies for Long COVID: A Narrative Review.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of infections to date and has led to a worldwide pandemic. Most patients had a complete recovery from the acute infection, however, a large number of the affected individuals experienced symptoms that persisted more than 3 months after diagnosis. These symptoms most commonly include fatigue, memory difficulties, brain fog, dyspnea, cough, and other less common ones such as headache, chest pain, paresthesias, mood changes, muscle pain, and weakness, skin rashes, and cardiac, endocrine, renal and hepatic manifestations. The treatment of this syndrome remains challenging. A multidisciplinary approach to address combinations of symptoms affecting multiple organ systems has been widely adopted. This narrative review aims to bridge the gap surrounding the broad treatment approaches by providing an overview of multidisciplinary management strategies for the most common long COVID conditions.

Proteoglycans of uterine fibroids and keloid scars: similarity in their proteoglycan composition.

Fibrosis is the formation of excess and abnormal fibrous connective tissue as a result of either a reparative or reactive process. A defining feature of connective tissue is its extracellular matrix, which provides structural support and also influences cellular activity. Two common human conditions that result from fibrosis are uterine fibroids (leiomyomas) and keloid scars. Because these conditions share a number of similarities and because their growth is due primarily to excessive extracellular matrix deposition, we compared the proteoglycans of uterine fibroids and keloid scars with corresponding normal tissues. Our analysis indicates that uterine fibroids and keloid scars contain higher amounts of glycosaminoglycans relative to normal myometrium and normal adult skin respectively. Proteoglycan composition is also different in the fibrotic tissues. Compared with unaffected tissues, uterine fibroids and keloid scars contain higher relative amounts of versican and lower relative amounts of decorin. There is also evidence for a higher level of versican catabolism in the fibrotic tissues compared with unaffected tissues. These qualitative and quantitative proteoglycan differences may play a role in the expansion of these fibroses and in their excessive matrix deposition and matrix disorganization, due to effects on cell proliferation, TGF (transforming growth factor)-ß signalling and/or collagen fibril formation.

The U.S. Food and Drug Administration, the mechanism of action, and other considerations for cell-based therapy candidates.

The focus of this Commentary is to introduce cell-based therapy in the context of how I believe the U.S. Food and Drug Administration (FDA) might establish criteria for the approval of clinical trials that could eventually lead to the final marketplace approval of these medically relevant, cell-based therapeutic products. It is important to emphasize that regulatory agencies have set up practices and procedures that are based on many years of evaluating pharmaceutically provided drugs. To consider cell-based therapies as single action drugs is inappropriate given the complexity of this technology. The regulatory agencies have been slowly reevaluating the criteria by which they allow clinical trials using cell-based therapies to proceed. This commentary focuses on a few key aspects of such considerations and provides suggestions for modifying the standard criteria.

Human Mesenchymal Stem Cell (hMSC) Donor Potency Selection for the "First in Cystic Fibrosis" Phase I Clinical Trial (CEASE-CF).

Human Mesenchymal Stem Cell (hMSC) immunotherapy has been shown to provide both anti-inflammatory and anti-microbial effectiveness in a variety of diseases. The clinical potency of hMSCs is based upon an initial direct hMSC effect on the pro-inflammatory and anti-microbial pathophysiology as well as sustained potency through orchestrating the host immunity to optimize the resolution of infection and tissue damage. Cystic fibrosis (CF) patients suffer from a lung disease characterized by excessive inflammation and chronic infection as well as a variety of other systemic anomalies associated with the consequences of abnormal cystic fibrosis transmembrane conductance regulator (CFTR) function. The application of hMSC immunotherapy to the CF clinical armamentarium is important even in the era of modulators when patients with an established disease still need anti-inflammatory and anti-microbial therapies. Additionally, people with CF mutations not addressed by current modulator resources need anti-inflammation and anti-infection management. Furthermore, hMSCs possess dynamic therapeutic properties, but the potency of their products is highly variable with respect to their anti-inflammatory and anti-microbial effects. Due to the variability of hMSC products, we utilized standardized in vitro and in vivo models to select hMSC donor preparations with the greatest potential for clinical efficacy. The models that were used recapitulate many of the pathophysiologic outcomes associated with CF. We applied this strategy in pursuit of identifying the optimal donor to utilize for the "First in CF" Phase I clinical trial of hMSCs as an immunotherapy and anti-microbial therapy for people with cystic fibrosis. The hMSCs screened in this study demonstrated significant diversity in antimicrobial and anti-inflammatory function using models which mimic some aspects of CF infection and inflammation. However, the variability in activity between in vitro potency and in vivo effectiveness continues to be refined. Future studies require and in-depth pursuit of hMSC molecular signatures that ultimately predict the capacity of hMSCs to function in the clinical setting.

A phase I study assessing the safety and tolerability of allogeneic mesenchymal stem cell infusion in adults with cystic fibrosis.

BACKGROUND: Mesenchymal stem cells are of particular interest in cystic fibrosis (CF) as a potential therapeutic. Data from pre-clinical studies suggest that allogeneic bone marrow-derived human mesenchymal stem cells (hMSCs) may provide a new therapeutic treatment for CF lung disease by attenuating pulmonary inflammation while decreasing bacterial growth and enhancing antibiotic efficacy. METHODS: Fifteen adults with CF were enrolled in a phase 1 dose-escalation trial of a single intravenous infusion of hMSCs derived from bone marrow aspirates obtained from a single pre-clinically validated healthy volunteer donor. The study employed a 3+3 dose escalation design with subjects receiving a single, intravenous dose of either 1×106, 3×106, or 5×106 hMSCs/kg. Subjects were monitored inpatient for 24 hours and by outpatient visits and telephone calls for 12 months after the infusion. Safety and tolerability were evaluated by monitoring symptoms, patient reported outcome questionnaires, adverse events (AEs), physical exam findings, spirometry, and analyses of safety laboratories. Preliminary evidence for potential efficacy using inflammatory markers in the blood and sputum were also evaluated. RESULTS: No dose-limiting toxicities, deaths or life-threatening adverse events were observed. Most AEs and serious adverse events (SAEs) were consistent with underlying CF. Vital signs, physical exam findings, spirometry and safety laboratory results showed no significant change from baseline. No trends over time were seen in serum or sputum inflammatory markers nor with clinical spirometry. CONCLUSION: Allogeneic hMSC intravenous infusions were safe and well-tolerated in this phase 1 study and warrant additional clinical testing as a potential therapeutic for CF lung disease.

Mesenchymal stem cells ameliorate inflammation in an experimental model of Crohn's disease via the mesentery.

Objective: Mesenchymal stem cells (MSCs) are novel therapeutics for treatment of Crohn's disease. However, their mechanism of action is unclear, especially in disease-relevant chronic models of inflammation. Thus, we used SAMP-1/YitFc, a chronic and spontaneous murine model of small intestinal inflammation, to study the therapeutic effect and mechanism of human bone marrow-derived MSCs (hMSC). Design: hMSC immunosuppressive potential was evaluated through in vitro mixed lymphocyte reaction, ELISA, macrophage co-culture, and RT-qPCR. Therapeutic efficacy and mechanism in SAMP were studied by stereomicroscopy, histopathology, MRI radiomics, flow cytometry, RT-qPCR, small animal imaging, and single-cell RNA sequencing (Sc-RNAseq). Results: hMSC dose-dependently inhibited naïve T lymphocyte proliferation in MLR via PGE 2 secretion and reprogrammed macrophages to an anti-inflammatory phenotype. hMSC promoted mucosal healing and immunologic response early after administration in SAMP model of chronic small intestinal inflammation when live hMSCs are present (until day 9) and resulted in complete response characterized by mucosal, histological, immunologic, and radiological healing by day 28 when no live hMSCs are present. hMSC mediate their effect via modulation of T cells and macrophages in the mesentery and mesenteric lymph nodes (mLN). Sc-RNAseq confirmed the anti-inflammatory phenotype of macrophages and identified macrophage efferocytosis of apoptotic hMSCs as a mechanism of action that explains their long-term efficacy. Conclusion: hMSCs result in healing and tissue regeneration in a chronic model of small intestinal inflammation. Despite being short-lived, exert long-term effects via macrophage reprogramming to an anti-inflammatory phenotype. Data Transparency Statement: Single-cell RNA transcriptome datasets are deposited in an online open access repository 'Figshare' (DOI: https://doi.org/10.6084/m9.figshare.21453936.v1 ).

Cell transplantation as an initiator of endogenous stem cell-based tissue repair.

PURPOSE OF REVIEW: The aim of this review is to summarize recent findings of endogenous cell-based tissue repair triggered by a multitude of approaches, especially stem cell therapy. RECENT FINDINGS: The emerging evidence has demonstrated that an important role of transplanted cells is to act as an initiator to trigger endogenous stem cell-mediated tissue repair after injury. Multiple approaches may orchestrate endogenous regeneration. The low efficacy of endogenous stem cells may be due to deficiency of cytokines to activate and induce endogenous stem cell homing, relatively insufficient endogenous stem cell pool, diseases or aging-related dysfunction of endogenous stem cells, and hostile microenvironments that limit the capacity of endogenous stem cells to repair damaged tissue. In certain situations, external stimulation and/or exogenous stem cells may be required to catalyze the repair. SUMMARY: Endogenous stem cells are playing an important role in tissue repair. An important role of transplanted cells is to act as an initiator to trigger endogenous stem cell-based tissue repair.