Umbilical cord-derived mesenchymal stem cell sheets transplanted subcutaneously enhance cell retention and survival more than dissociated stem cell injections.

BACKGROUND: Human umbilical cord-derived mesenchymal stem cell (hUC-MSC) sheets have recently attracted attention as an alternative approach to injected cell suspensions for stem cell therapy. However, cell engraftment and cytokine expression levels between hUC-MSC sheets and their cell suspensions in vivo have not yet been compared. This study compares hUC-MSC in vivo engraftment efficacy and cytokine expression for both hUC-MSC sheets and cell suspensions. METHODS: hUC-MSC sheets were prepared using temperature-responsive cell culture; two types of hUC-MSC suspensions were prepared, either by enzymatic treatment (trypsin) or by enzyme-free temperature reduction using temperature-responsive cell cultureware. hUC-MSC sheets and suspensions were transplanted subcutaneously into ICR mice through subcutaneous surgical placement and intravenous injection, respectively. hUC-MSC sheet engraftment after subcutaneous surgical transplantation was investigated by in vivo imaging while intravenously injected cell suspensions were analyzing using in vitro organ imaging. Cytokine levels in both transplant site tissues and blood were quantified by enzyme-linked immunosorbent assay. RESULTS: After subcutaneous transplant, hUC-MSC sheets exhibited longer engraftment duration than hUC-MSC suspensions. This was attributed to extracellular matrix (ECM) and cell-cell junctions retained in sheets but enzymatically altered in suspensions. hUC-MSC suspensions harvested using enzyme-free temperature reduction exhibited relatively long engraftment duration after intravenous injection compared to suspensions prepared using trypsin, as enzyme-free harvest preserved cellular ECM. High HGF and TGF-ß1 levels were observed in sheet-transplanted sites compared to hUC-MSC suspension sites. However, no differences in human cytokine levels in murine blood were detected, indicating that hUC-MSC sheets might exert local paracrine rather than endocrine effects. CONCLUSIONS: hUC-MSC sheet transplantation could be a more effective cell therapeutic approach due to enhanced engraftment and secretion of therapeutic cytokines over injected hUC-MSC suspensions.

Cellular Interactions in Cell Sheets Enhance Mesenchymal Stromal Cell Immunomodulatory Properties.

Immune-related applications of mesenchymal stromal cells (MSCs) in cell therapy seek to exploit immunomodulatory paracrine signaling pathways to reduce inflammation. A key MSC therapeutic challenge is reducing patient outcome variabilities attributed to insufficient engraftment/retention of injected heterogenous MSCs. To address this, we propose directly transplantable human single-cell-derived clonal bone marrow MSC (hcBMSC) sheets. Cell sheet technology is a scaffold-free tissue engineering strategy enabling scalable production of highly engraftable cell constructs retaining endogenous cell-cell and cell-matrix interactions, important to cell function. cBMSCs, as unique MSC subset populations, facilitate rational selection of therapeutically relevant MSC clones from donors. Here, we combine human cBMSCs with cell sheet technology, demonstrating cell sheet fabrication as a method to significantly upregulate expression of immunomodulatory molecules interleukin (IL)-10, indoleamine 2,3-dioxygenase (IDO-1), and prostaglandin E synthase 2 (PTGES2) across GMP-grade hcBMSC lines and whole human bone marrow-derived MSCs compared to respective conventional cell suspensions. When treated with carbenoxolone, a gap junction inhibitor, cell sheets downregulate IL-10 and IDO-1 expression, implicating functional roles for intercellular sheet interactions. Beyond producing directly transferable multicellular hcBMSC constructs, cell sheet technology amplifies hcBMSC expression of immunomodulatory factors important to therapeutic action. In addition, this work demonstrates the importance of cell-cell interactions as a tissue engineering design criterion to enhance consistent MSC functions.

Rapid and effective preparation of clonal bone marrow-derived mesenchymal stem/stromal cell sheets to reduce renal fibrosis.

Allogeneic "off-the-shelf" mesenchymal stem/stromal cell (MSC) therapy requires scalable, quality-controlled cell manufacturing and distribution systems to provide clinical-grade products using cryogenic cell banking. However, previous studies report impaired cell function associated with administering freeze-thawed MSCs as single cell suspensions, potentially compromising reliable therapeutic efficacy. Using long-term culture-adapted clinical-grade clonal human bone marrow MSCs (cBMSCs) in this study, we engineered cBMSC sheets in 24 h to provide rapid preparation. We then sought to determine the influence of cBMSC freeze-thawing on both in vitro production of pro-regenerative factors and in vivo ability to reduce renal fibrosis in a rat model compared to freshly harvested cBMSCs. Sheets from freeze-thawed cBMSCs sheets exhibited comparable in vitro protein production and gene expression of pro-regenerative factors [e.g., hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and interleukin 10 (IL-10)] to freshly harvested cBMSC sheets. Additionally, freeze-thawed cBMSC sheets successfully suppressed renal fibrosis in vivo in an established rat ischemia-reperfusion injury model. Despite previous studies reporting that freeze-thawed MSCs exhibit impaired cell functions compared to fresh MSC single cell suspensions, cell sheets engineered from freeze-thawed cBMSCs do not exhibit impaired cell functions, supporting critical steps toward future clinical translation of cBMSC-based kidney disease treatment.

Clinically Relevant Mesenchymal Stem/Stromal Cell Sheet Transplantation Method for Kidney Disease.

Chronic kidney disease (CKD) is the irreversible loss of nephron function, leading to a build-up of toxins, prolonged inflammation, and ultimately fibrosis. Currently, no effective therapies exist to treat CKD due to its complex pathophysiology. Mesenchymal stem/stromal cell (MSC) transplantation is a promising strategy to treat kidney diseases, and multiple clinical trials are currently ongoing. We previously demonstrated that rat bone marrow-derived MSC (BMSC) sheets transplanted onto surgically decapsulated kidney exert therapeutic effects that suppressed renal fibrosis progression based on enhanced vascularization. However, there are clinical concerns about kidney decapsulation such as impaired glomerular filtration rate and Na+ ion and H2O excretion, leading to kidney dysfunction. Therefore, for transitioning from basic research to translational research using cell sheet therapy for kidney disease, it is essential to develop a new cell sheet transplantation strategy without kidney decapsulation. Significantly, we employed cell sheets engineered from clinical-grade human clonal BMSC (cBMSC) and transplanted these onto intact renal capsule to evaluate their therapeutic ability in the rat ischemia-reperfusion injury (IRI) model. Histological analysis 1-day postsurgery showed that cBMSC sheets engrafted well onto intact renal capsule. Interestingly, some grafted cBMSCs migrated into the renal parenchyma. At 1-3 days postsurgery (acute stage), grafted cBMSC sheets prevented tubular epithelial cell injury. At 28 days postsurgery (chronic phase), we observed that grafted cBMSC sheets suppressed renal fibrosis in the rat IRI model. Taken together, engineered cBMSC sheet transplantation onto intact renal capsule suppresses tubular epithelial cell injury and renal fibrosis, supporting further development as a possible clinically relevant strategy. Impact statement Chronic kidney disease (CKD) produces irreversible loss of nephron function, leading to toxemia, prolonged inflammation, and ultimately kidney fibrosis. Currently, no therapies exist to effectively treat CKD due to its complex pathophysiology. Mesenchymal stem/stromal cells (MSCs) are widely known to secret therapeutic paracrine factors, which is expected to provide a new effective therapy for unmet medical needs. However, unsatisfied MSC quality and administration methods to patients limit their therapeutic effects. In this study, we engineered clonal bone marrow-derived MSC sheets and established clinically relevant cell sheet transplantation strategy to treat renal fibrosis, which would improve MSC treatment for kidney disease.

Interferon-Gamma Primed Human Clonal Mesenchymal Stromal Cell Sheets Exhibit Enhanced Immunosuppressive Function.

Mesenchymal stromal cells (MSCs) represent a promising treatment for immune-related diseases due to their diverse immunomodulatory paracrine functions. However, progress of culture-expanded MSCs is hindered by inconsistent cell function, poor localization, and insufficient retention when administered as suspended cell injections, thus placing spatiotemporal dosing constraints on therapeutic functions. To address these limitations, we introduce the combination of in vitro interferon-gamma (IFN-γ) priming, a key stimulator of MSC immunosuppressive potency, and thermoresponsive cultureware to harvest cultured MSCs as directly transplantable scaffold-free immunosuppressive cell sheets. Here, we demonstrate that MSC sheets produced with IFN-γ priming upregulate expression of immunosuppressive factors indoleamine 2,3-dioxygenase (IDO-1), interleukin-10 (IL-10), programmed death ligand-1 (PD-L1), and prostaglandin E2 (PGE2) in both dose- and duration-dependent manners. In addition, IFN-γ primed MSC sheets showed increased ability to inhibit T-cell proliferation via indirect and direct contact, specifically related to increased IDO-1 and PGE2 concentrations. Furthermore, this study's use of human clinical-grade single-cell-derived clonal bone marrow-derived MSCs, contributes to the future translatability and clinical relevancy of the produced sheets. Ultimately, these results present the combination of IFN-γ priming and MSC sheets as a new strategy to improve MSC-mediated treatment of localized inflammatory diseases.

Mesenchymal Stem Cell Sheet Centrifuge-Assisted Layering Augments Pro-Regenerative Cytokine Production.

A focal advantage of cell sheet technology has been as a scaffold-free three-dimensional (3D) cell delivery platform capable of sustained cell engraftment, survival, and reparative function. Recent evidence demonstrates that the intrinsic cell sheet 3D tissue-like microenvironment stimulates mesenchymal stem cell (MSC) paracrine factor production. In this capacity, cell sheets not only function as 3D cell delivery platforms, but also prime MSC therapeutic paracrine capacity. This study introduces a "cell sheet multilayering by centrifugation" strategy to non-invasively augment MSC paracrine factor production. Cell sheets fabricated by temperature-mediated harvest were first centrifuged as single layers using optimized conditions of rotational speed and time. Centrifugation enhanced cell physical and biochemical interactions related to intercellular communication and matrix interactions within the single cell sheet, upregulating MSC gene expression of connexin 43, integrin ß1, and laminin α5. Single cell sheet centrifugation triggered MSC functional enhancement, secreting higher concentrations of pro-regenerative cytokines vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10). Subsequent cell sheet stacking, and centrifugation generated cohesive, bilayer MSC sheets within 2 h, which could not be accomplished within 24 h by conventional layering methods. Conventional layering led to H1F-1α upregulation and increased cell death, indicating a hypoxic thickness limitation to this approach. Comparing centrifuged single and bilayer cell sheets revealed that layering increased VEGF production 10-fold, attributed to intercellular interactions at the layered sheet interface. The "MSC sheet multilayering by centrifugation" strategy described herein generates a 3D MSC-delivery platform with boosted therapeutic factor production capacity.

Specification of the haematopoietic stem cell lineage: From blood-fated mesodermal angioblasts to haemogenic endothelium.

Haematopoietic stem and progenitor cells emerge from specialized haemogenic endothelial cells in select vascular beds during embryonic development. Specification and commitment to the blood lineage, however, occur before endothelial cells are endowed with haemogenic competence, at the time of mesoderm patterning and production of endothelial cell progenitors (angioblasts). Whilst early blood cell fate specification has long been recognized, very little is known about the mechanisms that induce endothelial cell diversification and progressive acquisition of a blood identity by a subset of these cells. Here, we review the endothelial origin of the haematopoietic system and the complex developmental journey of blood-fated angioblasts. We discuss how recent technological advances will be instrumental to examine the diversity of the embryonic anatomical niches, signaling pathways and downstream epigenetic and transcriptional processes controlling endothelial cell heterogeneity and blood cell fate specification. Ultimately, this will give essential insights into the ontogeny of the cells giving rise to haematopoietic stem cells, that may aid in the development of novel strategies for their in vitro production for clinical purposes.

Human Mesenchymal Stem Cell Sheets Improve Uterine Incision Repair in a Rodent Hysterotomy Model.

OBJECTIVE: The study aimed to assess the feasibility of creating and transplanting human umbilical cord mesenchymal stem cell sheets applied to a rat model of hysterotomy, and additionally to determine benefits of human umbilical cord mesenchymal stem cell sheet transplantation in reducing uterine fibrosis and scarring. STUDY DESIGN: Human umbilical cord mesenchymal stem cell sheets are generated by culturing human umbilical cord mesenchymal stem cells on thermo-responsive cell culture plates. The temperature-sensitive property of these culture dishes facilitates normal cell culture in a thin contiguous layer and allows for reliable recovery of intact stem cell sheets without use of destructive proteolytic enzymes.We developed a rat hysterotomy model using nude rats. The rat uterus has two distinct horns: one horn provided a control/untreated scarring site, while the second horn was the cell sheet transplantation site.On day 14 following surgery, complete uteri were harvested and subjected to histologic evaluations of all hysterotomy sites. RESULTS: The stem cell sheet culture process yielded human umbilical cord mesenchymal stem cell sheets with surface area of approximately 1 cm2.Mean myometrial thickness in the cell sheet-transplanted group was 274 µm compared with 191 µm in the control group (p = 0.02). Mean fibrotic surface area in the human umbilical cord mesenchymal stem cell sheet-transplanted group was 95,861 µm2 compared with 129,185 µm2 in the control group. Compared with control horn sites, cell sheet-transplanted horns exhibited significantly smaller fibrotic-to-normal myometrium ratios (0.18 vs. 0.27, respectively, p = 0.029). Mean number of fibroblasts in cell sheet-transplanted horns was significantly smaller than the control horns (483 vs. 716/mm2, respectively, p = 0.001). CONCLUSION: Human umbilical cord mesenchymal stem cell sheet transplantation is feasible in a rat model of hysterotomy. Furthermore, use of stem cell sheets reduces fibroblast infiltration and uterine scar fibrotic tissue formation during hysterotomy healing, potentially mitigating risks of uterine scar formation. KEY POINTS: · Stem cell sheet transplanted to hysterotomy promotes myometrial regeneration and reduced fibrotic tissue formation.. · This study demonstrates the feasibility of using human umbilical cord mesenchymal stem cell sheets..

Trends in Articular Cartilage Tissue Engineering: 3D Mesenchymal Stem Cell Sheets as Candidates for Engineered Hyaline-Like Cartilage.

Articular cartilage defects represent an inciting factor for future osteoarthritis (OA) and degenerative joint disease progression. Despite multiple clinically available therapies that succeed in providing short term pain reduction and restoration of limited mobility, current treatments do not reliably regenerate native hyaline cartilage or halt cartilage degeneration at these defect sites. Novel therapeutics aimed at addressing limitations of current clinical cartilage regeneration therapies increasingly focus on allogeneic cells, specifically mesenchymal stem cells (MSCs), as potent, banked, and available cell sources that express chondrogenic lineage commitment capabilities. Innovative tissue engineering approaches employing allogeneic MSCs aim to develop three-dimensional (3D), chondrogenically differentiated constructs for direct and immediate replacement of hyaline cartilage, improve local site tissue integration, and optimize treatment outcomes. Among emerging tissue engineering technologies, advancements in cell sheet tissue engineering offer promising capabilities for achieving both in vitro hyaline-like differentiation and effective transplantation, based on controlled 3D cellular interactions and retained cellular adhesion molecules. This review focuses on 3D MSC-based tissue engineering approaches for fabricating "ready-to-use" hyaline-like cartilage constructs for future rapid in vivo regenerative cartilage therapies. We highlight current approaches and future directions regarding development of MSC-derived cartilage therapies, emphasizing cell sheet tissue engineering, with specific focus on regulating 3D cellular interactions for controlled chondrogenic differentiation and post-differentiation transplantation capabilities.

3D cell sheet structure augments mesenchymal stem cell cytokine production.

Mesenchymal stem cells (MSCs) secrete paracrine factors that play crucial roles during tissue regeneration. An increasing body of evidence suggests that this paracrine function is enhanced by MSC cultivation in three-dimensional (3D) tissue-like microenvironments. Toward this end, this study explored scaffold-free cell sheet technology as a new 3D platform. MSCs cultivated on temperature-responsive culture dishes to a confluent 2D monolayer were harvested by temperature reduction from 37 to 20 °C that induces a surface wettability transition from hydrophobic to hydrophilic. Release of culture-adherent tension induced spontaneous cell sheet contraction, reducing the diameter 2.4-fold, and increasing the thickness 8.0-fold to render a 3D tissue-like construct with a 36% increase in tissue volume. This 2D-to-3D transition reorganized MSC actin cytoskeleton from aligned to multidirectional, corresponding to a cell morphological change from elongated in 2D monolayers to rounded in 3D cell sheets. 3D culture increased MSC gene expression of cell interaction proteins, ß-catenin, integrin ß1, and connexin 43, and of pro-tissue regenerative cytokines, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10), and increased VEGF secretion per MSC 2.1-fold relative to 2D cultures. Together, these findings demonstrate that MSC therapeutic potency can be enhanced by 3D cell sheet tissue structure.

Strategies to address mesenchymal stem/stromal cell heterogeneity in immunomodulatory profiles to improve cell-based therapies.

Mesenchymal stromal cells (MSCs) have gained immense attention over the past two decades due to their multipotent differentiation potential and pro-regenerative and immunomodulatory cytokine secretory profiles. Their ability to modulate the host immune system and promote tolerance has prompted several allogeneic and autologous hMSC-based clinical trials for the treatment of graft-versus-host disease and several other immune-induced disorders. However, clinical success beyond safety is still controversial and highly variable, with inconclusive therapeutic benefits and little mechanistic explanation. This clinical variability has been broadly attributed to inconsistent MSC sourcing, phenotypic characterization, variable potency, and non-standard isolation protocols, leading to functional heterogeneity among administered MSCs. Homogeneous MSC populations are proposed to yield more predictable, reliable biological responses and clinically meaningful properties relevant to cell-based therapies. Limited comparisons of heterogeneous MSCs with homogenous MSCs are reported. This review addresses this gap in the literature with a critical analysis of strategies aimed at decreasing MSC heterogeneity concerning their reported immunomodulatory profiles. STATEMENT OF SIGNIFICANCE: This review collates, summarizes, and critically analyzes published strategies that seek to improve homogeneity in immunomodulatory functioning MSC populations intended as cell therapies to treat immune-based disorders, such as graft-vs-host-disease. No such review for MSC therapies, immunomodulatory profiles and cell heterogeneity analysis is published. Since MSCs represent the most clinically studied experimental cell therapy platform globally for which there remains no US domestic marketing approval, insights into MSC challenges in therapeutic product development are imperative to providing solutions for immunomodulatory variabilities.

Allogeneic mesenchymal stem cell sheet therapy: A new frontier in drug delivery systems.

The evolution of drug discovery exploded in the early 20th century with the advent of critical scientific advancements in organic chemistry, chemical analysis, and purification. Early drug generations focused largely on symptom control and pain management, effective targets for small-molecule drugs. Recently, the attention in drug discovery has shifted to pursuit of radical cures. Cell therapy presents the ideal attributes of a promising new drug, targeting specific tissues based on chemotactic cues and modulating secretion of instructive regenerative molecules in response to dynamic signaling from disease environments. To actuate the therapeutic potential of cell therapy toward worldwide clinical use, cell delivery methods that can effectively localize and engraft mesenchymal stem cells (MSCs) with high disease-site fidelity and enable dynamic MSC bioactive function are paramount. In this review, we discuss the evolution of cell therapies with a focus on stem cell advantages, as well as the limitations to these therapies. This review aims to introduce cell sheet technology as a breakthrough cell therapy with demonstrated therapeutic success across indications for heart, liver, and kidney tissue regeneration. Opportunities and anticipated clinical impacts of cell sheet technology using MSCs are discussed.

Innovative Molecular and Cellular Therapeutics in Cleft Palate Tissue Engineering.

Clefts of the lip and/or palate are the most prevalent orofacial birth defects occurring in about 1:700 live human births worldwide. Early postnatal surgical interventions are extensive and staged to bring about optimal growth and fusion of palatal shelves. Severe cleft defects pose a challenge to correct with surgery alone, resulting in complications and sequelae requiring life-long, multidisciplinary care. Advances made in materials science innovation, including scaffold-based delivery systems for precision tissue engineering, now offer new avenues for stimulating bone formation at the site of surgical correction for palatal clefts. In this study, we review the present scientific literature on key developmental events that can go awry in palate development and the common surgical practices and challenges faced in correcting cleft defects. How key osteoinductive pathways implicated in palatogenesis inform the design and optimization of constructs for cleft palate correction is discussed within the context of translation to humans. Finally, we highlight new osteogenic agents and innovative delivery systems with the potential to be adopted in engineering-based therapeutic approaches for the correction of palatal defects. Impact statement Tissue-engineered scaffolds supplemented with osteogenic growth factors have attractive, largely unexplored possibilities to modulate molecular signaling networks relevant to driving palatogenesis in the context of congenital anomalies (e.g., cleft palate). Constructs that address this need may obviate current use of autologous bone grafts, thereby avoiding donor-site morbidity and other regenerative challenges in patients afflicted with palatal clefts. Combinations of biomaterials and drug delivery of diverse regenerative cues and biologics are currently transforming strategies exploited by engineers, scientists, and clinicians for palatal cleft repair.

Fabrication of hyaline-like cartilage constructs using mesenchymal stem cell sheets.

Cell and tissue engineering approaches for articular cartilage regeneration increasingly focus on mesenchymal stem cells (MSCs) as allogeneic cell sources, based on availability and innate chondrogenic potential. Many MSCs exhibit chondrogenic potential as three-dimensional (3D) cultures (i.e. pellets and seeded biomaterial scaffolds) in vitro; however, these constructs present engraftment, biocompatibility, and cell functionality limitations in vivo. Cell sheet technology maintains cell functionality as scaffold-free constructs while enabling direct cell transplantation from in vitro culture to targeted sites in vivo. The present study aims to develop transplantable hyaline-like cartilage constructs by stimulating MSC chondrogenic differentiation as cell sheets. To achieve this goal, 3D MSC sheets are prepared, exploiting spontaneous post-detachment cell sheet contraction, and chondrogenically induced. Results support 3D MSC sheets' chondrogenic differentiation to hyaline cartilage in vitro via post-contraction cytoskeletal reorganization and structural transformations. These 3D cell sheets' initial thickness and cellular densities may also modulate MSC-derived chondrocyte hypertrophy in vitro. Furthermore, chondrogenically differentiated cell sheets adhere directly to cartilage surfaces via retention of adhesion molecules while maintaining the cell sheets' characteristics. Together, these data support the utility of cell sheet technology for fabricating scaffold-free, hyaline-like cartilage constructs from MSCs for future transplantable articular cartilage regeneration therapies.

Novel therapies using cell sheets engineered from allogeneic mesenchymal stem/stromal cells.

Mesenchymal stem/stromal cells (MSCs) have long been recognized to help regenerate tissues, by exploiting their intrinsic potentials for differentiation and secretion of therapeutic paracrine factors together with feasibility for cell banking. These unique MSC properties are attractive to provide effective new cell-based therapies for unmet medical needs. Currently, the infusion of suspended MSCs is accepted as a promising therapy to treat systemic inflammatory diseases. However, low cell engraftment/retention in target organs and off-target entrapment using conventional cell infusion must be improved to provide reliable localized disease treatments. Cell sheet technology offers an alternative: three-dimensional (3D) tissue-like structures can be harvested from culture using mild temperature reduction, and transplanted directly onto target tissue sites without suturing, yielding stable cell engraftment and prolonged cell retention in situ without off-target losses. Engineered MSC sheets directly address two major cell therapy strategies based on their therapeutic benefits: (1) tissue replacements based on mult-ilineage differentiation capacities, focusing on cartilage regeneration in this review, and (2) enhancement of tissue recovery via paracrine signaling, employing their various secreted cytokines to promote neovascularization. MSCs also have production benefits as a promising allogeneic cell source by exploiting their reliable proliferative capacity to facilitate expansion and sustainable cell banking for off-the-shelf therapies. This article reviews the advantages of both MSCs as allogeneic cell sources in contrast with autologous cell sources, and allogeneic MSC sheets engineered on thermo-responsive cell dishes as determined in basic studies and clinical achievements, indicating promise to provide robust new cell therapies to future patients.

Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

Activin receptor-like kinase 1 (ALK1)-mediated endothelial cell signalling in response to bone morphogenetic protein 9 (BMP9) and BMP10 is of significant importance in cardiovascular disease and cancer. However, detailed molecular mechanisms of ALK1-mediated signalling remain unclear. Here, we report crystal structures of the BMP10:ALK1 complex at 2.3 Å and the prodomain-bound BMP9:ALK1 complex at 3.3 Å. Structural analyses reveal a tripartite recognition mechanism that defines BMP9 and BMP10 specificity for ALK1, and predict that crossveinless 2 is not an inhibitor of BMP9, which is confirmed by experimental evidence. Introduction of BMP10-specific residues into BMP9 yields BMP10-like ligands with diminished signalling activity in C2C12 cells, validating the tripartite mechanism. The loss of osteogenic signalling in C2C12 does not translate into non-osteogenic activity in vivo and BMP10 also induces bone-formation. Collectively, these data provide insight into ALK1-mediated BMP9 and BMP10 signalling, facilitating therapeutic targeting of this important pathway.

Stable cell adhesion affects mesenchymal stem cell sheet fabrication: Effects of fetal bovine serum and human platelet lysate.

Cell sheet technology exploits temperature responsive cell culture dishes (TRCDs) as versatile cell harvesting methods to yield contiguous cell monolayers robustly held together by cell-cell junctions, receptors, and endogenous extracellular matrix. More than 15 years of clinical data using autologous-sourced cell sheets demonstrate enhanced therapeutic properties through increased cell retention at target tissue sites. Recently, several preclinical studies have also been reported using mesenchymal stem cell (MSC) sheets in wound healing, cardiac ischemia therapies, and pancreatic regeneration. However, optimized MSC sheet fabrication conditions have not yet been reported. In this study, we identified specific conditions for reliable human MSC sheet fabrication by comparing cell growth media supplements (fetal bovine serum [FBS] and human platelet lysate [hPL]). Human umbilical cord-derived MSCs cultured in FBS and hPL exhibit different actin cytoskeletal structures related to their cell morphologies and adhesion. MSCs cultured in FBS media showed stable cell adhesion on TRCDs with flattened cell shapes and aligned actin cytoskeletal structure. This stable cell adhesion enables production of consistent MSC cell sheets, with controlled cell sheet detachment. Conversely, cell sheet fabrication in hPL media exhibits poor reproducibility being more sensitive to temperature- and culture time-induced release due to weak cell adhesion. These findings suggest that stable MSC adhesion to TRCDs is important to reliable MSC sheet fabrication methods and that MSC growth media supplementation directly affects cell adhesion during culture.

Phenotypic traits of mesenchymal stem cell sheets fabricated by temperature-responsive cell culture plate: structural characteristics of MSC sheets.

BACKGROUND: In most stem cell therapy strategies reported to date, stem cells are introduced to damaged tissue sites to repair and regenerate the original tissue structure and function. MSC therapeutic efficacies are inconsistent, largely attributed to transplanted MSC difficulties both in engrafting at tissue sites and in retaining their therapeutic functions from suspension formulations. MSC functional components, including cell adhesion and cell-cell junction proteins, and ECM that contribute to essential cellular therapeutic effects, are damaged or removed by proteolytic enzymes used in stem cell harvesting strategies from culture. To overcome these limitations, methods to harvest and transplant cells without disrupting critical stem cell functions are required. Cell sheet technology, exploiting temperature-responsive cell culture surfaces, permits cell harvest without cell protein damage. This study is focused on phenotypic traits of MSC sheets structurally and functionally to understand therapeutic benefits of cell sheets. METHODS/RESULTS: This study verified cleaved cellular proteins (vinculin, fibronectin, laminin, integrin ß-1, and connexin 43) and increased apoptotic cell death produced under standard trypsin harvesting treatment in a time-dependent manner. However, MSC sheets produced without trypsin using only temperature-controlled sheet harvest from culture plastic exhibited intact cellular structures. Also, MSCs harvested using enzymatic treatment (i.e., chemical disruption) showed higher pYAP expression compared to MSC sheets. CONCLUSION: Retention of cellular structures such as ECM, cell-cell junctions, and cell-ECM junctions is correlated with human umbilical cord mesenchymal stem cell (hUC-MSC) survival after detachment from cell culture surfaces. Retaining these proteins intact in MSC cultures using cell sheet technology is proposed to enhance stem cell survival and their function in stem cell-based therapy.

Human mesenchymal stem cell sheets in xeno-free media for possible allogenic applications.

Cell-based therapies are increasingly focused on allogeneic stem cell sources because of several advantages in eliminating donor variability (e.g., aging and disease pathophysiology) affecting stem cell quality and in cell-banked sourcing of healthy donors to enable "off-the-shelf" products. However, allogeneic cell therapy is limited by host patient immunologic competence and inconsistent performance due to cell delivery methods. To address allogeneic cell therapy limitations, this study developed a new allogeneic stem cell sheet using human umbilical cord mesenchymal stem cells (hUC-MSC) that present low antigenicity (i.e., major histocompatibility complex, MHC). Optimal conditions including cell density, passage number, and culture time were examined to fabricate reliable hUC-MSC sheets. MHC II antigens correlated to alloimmune rejection were barely expressed in hUC-MSC sheets compared to other comparator MSC sheets (hBMSC and hADSC). hUC-MSC sheets easily graft spontaneously onto subcutaneous tissue in immune-deficient mice within 10 minutes of placement. No sutures are required to secure sheets to tissue because sheet extracellular matrix (ECM) actively facilitates cell-target tissue adhesion. At 10 days post-transplantation, hUC-MSC sheets remain on ectopic target tissue sites and exhibit new blood vessel formation. Furthermore, implanted hUC-MSC sheets secrete human HGF continuously to the murine target tissue. hUC-MSC sheets described here should provide new insights for improving allogenic cell-based therapies.

Recommendations for design and conduct of preclinical in vivo studies of orthopedic device-related infection.

Orthopedic device-related infection (ODRI), including both fracture-related infection (FRI) and periprosthetic joint infection (PJI), remain among the most challenging complications in orthopedic and musculoskeletal trauma surgery. ODRI has been convincingly shown to delay healing, worsen functional outcome and incur significant socio-economic costs. To address this clinical problem, ever more sophisticated technologies targeting the prevention and/or treatment of ODRI are being developed and tested in vitro and in vivo. Among the most commonly described innovations are antimicrobial-coated orthopedic devices, antimicrobial-loaded bone cements and void fillers, and dual osteo-inductive/antimicrobial biomaterials. Unfortunately, translation of these technologies to the clinic has been limited, at least partially due to the challenging and still evolving regulatory environment for antimicrobial drug-device combination products, and a lack of clarity in the burden of proof required in preclinical studies. Preclinical in vivo testing (i.e. animal studies) represents a critical phase of the multidisciplinary effort to design, produce and reliably test both safety and efficacy of any new antimicrobial device. Nonetheless, current in vivo testing protocols, procedures, models, and assessments are highly disparate, irregularly conducted and reported, and without standardization and validation. The purpose of the present opinion piece is to discuss best practices in preclinical in vivo testing of antimicrobial interventions targeting ODRI. By sharing these experience-driven views, we aim to aid others in conducting such studies both for fundamental biomedical research, but also for regulatory and clinical evaluation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:271-287, 2019.