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.

Engineered Bone Marrow Stem Cell-Sheets Alleviate Renal Damage in a Rat Chronic Glomerulonephritis Model.

Although mesenchymal stem cell (MSC)-based regenerative therapy is being developed for the treatment of kidney diseases, cell delivery and engraftment still need to be improved. Cell sheet technology has been developed as a new cell delivery method, to recover cells as a sheet form retaining intrinsic cell adhesion proteins, which promotes its transplantation efficiency to the target tissue. We thus hypothesized that MSC sheets would therapeutically reduce kidney disease with high transplantation efficiency. When the chronic glomerulonephritis was induced by two injections of the anti-Thy 1.1 antibody (OX-7) in rats, the therapeutic efficacy of rat bone marrow stem cell (rBMSC) sheet transplantation was evaluated. The rBMSC-sheets were prepared using the temperature-responsive cell-culture surfaces and transplanted as patches onto the surface of two kidneys of each rat at 24 h after the first injection of OX-7. At 4 weeks, retention of the transplanted MSC-sheets was confirmed, and the animals with MSC-sheets showed significant reductions in proteinuria, glomerular staining for extracellular matrix protein, and renal production of TGFß1, PAI-1, collagen I, and fibronectin. The treatment also ameliorated podocyte and renal tubular injury, as evidenced by a reversal in the reductions of WT-1, podocin, and nephrin and by renal overexpression of KIM-1 and NGAL. Furthermore, the treatment enhanced gene expression of regenerative factors, and IL-10, Bcl-2, and HO-1 mRNA levels, but reduced TSP-1 levels, NF-kB, and NAPDH oxidase production in the kidney. These results strongly support our hypothesis that MSC-sheets facilitated MSC transplantation and function, and effectively retarded progressive renal fibrosis via paracrine actions on anti-cellular inflammation, oxidative stress, and apoptosis and promoted regeneration.

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.

Safety and efficacy of human juvenile chondrocyte-derived cell sheets for osteochondral defect treatment.

Knee cartilage does not regenerate spontaneously after injury, and a gold standard regenerative treatment algorithm has not been established. This study demonstrates preclinical safety and efficacy of scaffold-free, human juvenile cartilage-derived-chondrocyte (JCC) sheets produced from routine surgical discards using thermo-responsive cultureware. JCCs exhibit stable and high growth potential in vitro over passage 10, supporting possibilities for scale-up to mass production for commercialization. JCC sheets contain highly viable, densely packed cells, show no anchorage-independent cell growth, express mesenchymal surface markers, and lack MHC II expression. In nude rat focal osteochondral defect models, stable neocartilage formation was observed at 4 weeks by JCC sheet transplantation without abnormal tissue growth over 24 weeks in contrast to the nontreatment group showing no spontaneous cartilage repair. Regenerated cartilage was safranin-O positive, contained type II collagen, aggrecan, and human vimentin, and lacked type I collagen, indicating that the hyaline-like neocartilage formed originates from transplanted JCC sheets rather than host-derived cells. This study demonstrates the safety of JCC sheets and stable hyaline cartilage formation with engineered JCC sheets utilizing a sustainable tissue supply. Cost-benefit and scaling issues for sheet fabrication and use support feasibility of this JCC sheet strategy in clinical cartilage repair.

The Amot/integrin protein complex transmits mechanical forces required for vascular expansion.

Vascular development is a complex multistep process involving the coordination of cellular functions such as migration, proliferation, and differentiation. How mechanical forces generated by cells and transmission of these physical forces control vascular development is poorly understood. Using an endothelial-specific genetic model in mice, we show that deletion of the scaffold protein Angiomotin (Amot) inhibits migration and expansion of the physiological and pathological vascular network. We further show that Amot is required for tip cell migration and the extension of cellular filopodia. Exploiting in vivo and in vitro molecular approaches, we show that Amot binds Talin and is essential for relaying forces between fibronectin and the cytoskeleton. Finally, we provide evidence that Amot is an important component of the endothelial integrin adhesome and propose that Amot integrates spatial cues from the extracellular matrix to form a functional vascular network.

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.

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.

Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells.

Remodeling of epithelial cells and basement membranes in a corneal deficiency model with long-term follow-up.

The ocular surface consists of the cornea, conjunctiva, and the limbus that is located in the transitional zone between the cornea and conjunctiva. The corneal epithelial cells are generated through the mitosis of corneal epithelial stem cells in the limbus. This study investigated a rabbit corneal deficiency model prepared by the surgical removal of the corneal and limbal epithelia, which express cytokeratin 12 (K12). After the surgery, K13-expressing conjunctival epithelium migrated onto the corneal surface and completely covered the surface, leading to neovascularization and corneal opacification. However, at 24 and 48 weeks after the surgery, K12-expressing cornea-like cells reappeared on the model ocular surface. These cells formed an island surrounded by invaded conjunctiva and were isolated from the limbus. Interestingly, in the 24-week model surface, α1(IV) and α2(IV) collagen chains, which are normally found in the basement membrane of the native limbus and conjunctiva, and not in the cornea, were continuously deposited throughout the entire basement membrane, including the basement membrane under cornea-like cells. By contrast, in the 48-week model surface, α1(IV) and α2(IV) collagen chains were absent from the basement membrane beneath the central part of cornea-like cells and were localized below the invaded conjunctiva and the transitional zone between cornea-like cells and the invaded conjunctiva, which had similar distribution to the native ocular basement membrane. Moreover, K12, K14, p63, vimentin, and α1(IV) and α2(IV) collagen chains, which are colocalized in the native limbus, were all present at the transitional zone of the 48-week model surface. Therefore, a limbus-like structure appeared to be reconstructed on the surface of the 48-week model as a stem cell niche. This study should aid in the understanding of human corneal deficiency, the correlation between the epithelial cell phenotype and the composition of the basement membrane, and the epithelial stem cell niche.