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.

Long-term efficacy and safety of intravenous injection of clonal mesenchymal stem cells derived from bone marrow in five adults with moderate to severe atopic dermatitis.

Atopic dermatitis is a chronic and relapsing inflammatory skin disease that is treated with immunosuppressants. However, long-term use of immunosuppressants may cause toxicity and severe side-effects. To confirm the long-term efficacy and safety of clonal mesenchymal stem cell therapy, we performed investigator-initiated clinical trials and long-term observation in five adult patients with moderate to severe atopic dermatitis that was refractory to conventional treatments. The clinical response assessment values such as Eczema Area and Severity Index (EASI) improved significantly at 16 weeks, and 80% (4/5) of the patients achieved EASI-50 after one or two treatment cycles. Patients were observed for long-term efficacy and safety for an average of 38 weeks (range, 16-86) and showed no serious side-effects. Among the cytokines tested, CCL-17, interleukin (IL)-13, and IL-22 significantly decreased at the end-point of the five participants, two patients who maintained good clinical response over 84 weeks showed increased IL-17 cytokine levels in the blood.

A Novel Immunomodulatory Mechanism Dependent on Acetylcholine Secreted by Human Bone Marrow-derived Mesenchymal Stem Cells.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) are used to treat autoimmune or inflammatory diseases. Our aim was to determine the immunomodulatory mechanisms elicited by MSCs during inflammation. METHODS AND RESULTS: We cocultured MSCs with peripheral blood mononuclear cells for a mixed lymphocyte reaction or stimulated them by phytohemagglutinin. Morphological changes of MSCs and secretion of acetylcholine (ACh) from MSCs were measured. The effects of an ACh antagonist and ACh agonist on lymphocyte proliferation and proinflammatory-cytokine production were determined. The inflammatory milieu created by immune-cell activation caused MSCs to adopt a neuronlike phenotype and induced them to release ACh. Additionally, nicotinic acetylcholine receptors (nAChRs) were upregulated in activated peripheral blood mononuclear cells. We observed that ACh bound to nAChR on activated immune cells and led to the inhibition of lymphocyte proliferation and of proinflammatory-cytokine production. MSC-mediated immunosuppression through ACh activity was reversed by an ACh antagonist called α-bungarotoxin, and lymphocyte proliferation was inhibited by an ACh agonist, ACh chloride. CONCLUSIONS: Our findings point to a novel immunomodulatory mechanism in which ACh secreted by MSCs under inflammatory conditions might modulate immune cells. This study may provide a novel method for the treatment of autoimmune diseases by means of MSCs.

Comparative study on metabolite level in tissue-specific human mesenchymal stem cells by an ultra-performance liquid chromatography quadrupole time of flight mass spectrometry.

Mesenchymal stem cells (MSCs) are a promising therapeutic option for cell-based therapy due to their immunomodulatory and regenerative properties. They can be isolated from various adult tissues, including bone marrow, fat, dental tissue, and glandular tissue. Although they share common characteristics, little is known about the biological differences between MSC populations derived from different tissues. In this study, we used MS to compare the endogenous metabolite level in the human MSCs originating from the bone marrow, adipose tissue, periodontal ligaments, and salivary glands. Using an optimized metabolomics technique, we verified that human MSCs exhibit differences in the endogenous metabolite level depending on their source material, while the multivariate analysis showed that 5 lysophosphatidylcholines and 3 lysophosphatidylethanolamines can serve as markers for the discrimination between MSC sources and may be related to differences in their differentiation capacity. These results may significantly contribute to further mechanistic studies on the MSCs and provide novel insights into the properties and optimal usage of MSCs from different tissues.

Manufacture of Clinical-Grade Human Clonal Mesenchymal Stem Cell Products from Single Colony Forming Unit-Derived Colonies Based on the Subfractionation Culturing Method.

Stem cell products derived from mesenchymal stem cells (MSCs) have been widely used in clinical trials, and a few products have been already commercialized. However, the therapeutic effects of clinical-grade MSCs are still controversial owing to mixed results from recent clinical trials. A potential solution to overcome this hurdle may be to use clonal stem cells as the starting cell material to increase the homogeneity of the final stem cell products. We have previously developed an alternative isolation and culture protocol for establishing a population of clonal MSCs (cMSCs) from single colony forming unit (CFU)-derived colonies. In this study, we established a good manufacturing practice (GMP)-compatible procedure for the clinical-grade production of human bone marrow-derived cMSCs based on the subfractionation culturing method. We optimized the culture procedures to expand and obtain a clonal population of final MSC products from single CFU-derived colonies in a GMP facility. The characterization results of the final cMSC products met our preset criteria. Animal toxicity tests were performed in a good laboratory practice facility, and showed no toxicity or tumor formation in vivo. These tests include single injection toxicity, multiple injection toxicity, biodistribution analysis, and tumorigenicity tests in vivo. No chromosomal abnormalities were detected by in situ karyotyping using oligo-fluorescence in situ hydridization (oligo-FISH), providing evidence of genetic stability of the clinical-grade cMSC products. The manufacture and quality control results indicated that our GMP methodology could produce sufficient clonal population of MSC products from a small amount of bone marrow aspirate to treat a number of patients.

Biomimetic chimeric peptide-tethered hydrogels for human mesenchymal stem cell delivery.

Here, we report a chimeric peptide-tethered fibrin hydrogel scaffold for delivery of human mesenchymal stem cells (hMSC). Osteopontin-derived peptide (OP) was used as an hMSC-tethering moiety. OP showed hMSC adhesion properties and enhanced hMSC proliferation. A natural fibrin-binding protein-derived peptide (FBP) was tested for its ability to tether hMSC to the fibrin gel matrix. FBP loading on fibrin gels was 8.2-fold higher than that of a scrambled peptide (scFBP). FBP-loaded fibrin gels were retained at injection sites longer than scFBP-loaded fibrin gels, showing a 15.9-fold higher photon intensity of fluorescent FBP-grafted fibrin gels than fluorescent scFBP-loaded fibrin gels 48 h after injection. On the basis of the fibrin gel-binding properties of FBP and the hMSC-binding and proliferation-supporting properties of OP, we constructed chimeric peptides containing FBP and OP linked with a spacer (FBPsOP). Four days after transplantation, the survival of hMSC in FBPsOP-grafted fibrin gels was 3.9-fold higher than hMSC in fibrin gels alone. Our results suggest the potential of FBPsOP-grafted fibrin gels as a bioactive delivery system for enhanced survival of stem cells.

Pharmacokinetics and in vivo fate of intra-articularly transplanted human bone marrow-derived clonal mesenchymal stem cells.

In this study, we report the pharmacokinetics and in vivo fate of intra-articularly transplanted human mesenchymal stem cells (MSCs) in comparison with those of intravenously administered cells. Bone marrow-derived human clonal mesenchymal stem cells (hcMSCs) were transplanted to nude mice through intravenous or intra-articular routes. The numbers of hcMSCs in blood and tissue samples were measured by the quantitative real-time-polymerase chain reaction (qPCR) with human Alu (hAlu) as a detection marker. Following intra-articular transplantation, the blood levels of hcMSCs peaked 8 h postdose and gradually diminished, showing a 95-fold higher mean residence time than hcMSCs delivered through the intravenous route. Unlike intravenously administered hcMSCs, intra-articularly injected hcMSCs were mainly retained at injection joint sites where their levels 8 h postdose were 116-fold higher than those in muscle tissues. Regardless of injection routes, biodistribution patterns did not significantly differ between normal and osteoarthritis-induced mice. Quantitative analysis using hAlu-specific qPCR revealed that hcMSC levels in joint tissues were significantly higher than those in muscle tissues 120 days postdose. These dramatic differences in kinetic behavior and fate of intra-articularly transplanted hcMSCs compared with intravenously administered hcMSCs may provide insights useful for the development of human MSCs for arthritis therapeutics.

Non-invasive characterization of the adipogenic differentiation of human bone marrow-derived mesenchymal stromal cells by HS-SPME/GC-MS.

A non-invasive method to characterize human mesenchymal stromal cells during adipogenic differentiation was developed for the first time. Seven fatty acid methyl esters (FAMEs), including methyl laurate, methyl myristate, methyl palmitate, methyl linoleate, methyl oleate, methyl elaidate and methyl stearate, were used for characterizing adipogenic differentiation using headspace solid-phase microextraction (HS-SPME) which is a very simple and non-invasive method for the extraction of volatile compounds. Glassware was used for culturing mesenchymal stromal cells rather than the common plasticware to minimize contamination by volatile impurities. The optimal SPME fiber was selected by comparing diverse fibers containing two pure liquid polymers (PDMS and PA) and two porous solids (PDMS/DVB and CAR/PDMS). Using optimized procedures, we discovered that seven FAMEs were only detected in adipogenic differentiated mesenchymal stromal cells and not in the mesenchymal stromal cells before differentiation. These data could support the quality control of clinical mesenchymal stromal cell culture in the pharmaceutical industry in addition to the development of many clinical applications using mesenchymal stromal cells.

Isolation of adipose-derived stem cells by using a subfractionation culturing method.

OBJECTIVE: Adipose-derived stem cells (ASCs) isolated from subcutaneous adipose tissue have been tested in clinical trials. However, ASCs isolated by enzyme digestion and centrifugation are heterogeneous and exhibit wide variation in regenerative potential and clinical outcomes. Therefore, we developed a new method for isolating clonal ASCs (cASCs) that does not use enzyme digestion or centrifugation steps. RESEARCH DESIGN AND METHODS: In addition to cell surface markers and differentiation potential, we compared the mitogenic, paracrine and hair growth-promoting effects of ASCs isolated by the gradient centrifugation method (GCM) or by the new subfractionation culturing method (SCM). RESULTS: We selected three cASCs isolated by SCM that showed high rates of proliferation. The cell surface markers expressed by ASCs isolated by GCM or SCM were very similar, and SCM-isolated ASCs could potentially differentiate into different cell lineages. However, cASC lines exhibited better mitogenic and paracrine effects than ASCs isolated by GCM. The expression of Diras3, Myb, Cdca7, Mki67, Rrm2, Cdk1 and Ccna2, which may play a key role in cASC proliferation, was upregulated in cASCs. In addition, cASCs exhibited enhanced hair growth-promoting effects in dermal papilla cells and animal experiments. CONCLUSIONS: SCM generates a highly homogeneous population of ASCs via a simple and effective procedure that can be used in therapeutic settings.

Molecular Characterization of Neurally Differentiated Human Bone Marrow-derived Clonal Mesenchymal Stem Cells.

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent, with the ability to differentiate into different cell types. Additionally, the immunomodulatory activity of MSCs can downregulate inflammatory responses. The use of MSCs to repair injured tissues and treat inflammation, including in neuroimmune diseases, has been extensively explored. Although MSCs have emerged as a promising resource for the treatment of neuroimmune diseases, attempts to define the molecular properties of MSCs have been limited by the heterogeneity of MSC populations. We recently developed a new method, the subfractionation culturing method, to isolate homogeneous human clonal MSCs (hcMSCs). The hcMSCs were able to differentiate into fat, cartilage, bone, neuroglia, and liver cell types. In this study, to better understand the properties of neurally differentiated MSCs, gene expression in highly homogeneous hcMSCs was analyzed. Neural differentiation of hcMSCs was induced for 14 days. Thereafter, RNA and genomic DNA was isolated and subjected to microarray analysis and DNA methylation array analysis, respectively. We correlated the transcriptome of hcMSCs during neural differentiation with the DNA methylation status. Here, we describe and discuss the gene expression profile of neurally differentiated hcMSCs. These findings will expand our understanding of the molecular properties of MSCs and contribute to the development of cell therapy for neuroimmune diseases.

Senescing human bone-marrow-derived clonal mesenchymal stem cells have altered lysophospholipid composition and functionality.

Mesenchymal stem cells (MSCs) have been used in a wide range of research and clinical studies because MSCs do not have any ethical issues and have the advantage of low carcinogenicity due to their limited proliferation. However, because only a small number of MSCs can be obtained from the bone marrow, ex vivo amplification is inevitably required. For that reason, this study was conducted to acquire the metabolic information to examine and control the changes in the activities and differentiation potency of MSCs during the ex vivo culture process. Endogenous metabolites of human bone-marrow-derived clonal MSCs (hcMSCs) during cellular senescence were profiled by ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/QTOFMS). To select significant metabolites, we used the linear mixed effects model having fixed effects for batch and time (passage) and random effects for metabolites, determining the mean using a t test and the standard deviation using an F test. We used structural analysis with representative standards and spectrum patterns with different collision energies to distinctly identify eight metabolites with altered expression during senescence as types of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), such as LPC 16:0 and LPE 22:4. The present study revealed changes in endogenous metabolites and mechanisms due to senescence.

Mesenchymal Stem Cell Lines Isolated by Different Isolation Methods Show Variations in the Regulation of Graft-versus-host Disease.

Since the discovery of the immunomodulation property of mesenchymal stem cells (MSCs) about a decade ago, it has been extensively investigated whether MSCs can be used for the treatment of immune-related diseases, such as graft-versus-host disease (GvHD). However, how to evaluate the efficacy of human MSCs for the clinical trial is still unclear. We used an MHC-mismatched model of GvHD (B6 into BALB/c). Surprisingly, the administration of the human MSCs (hMSCs) could reduce the GvHD-related mortality of the mouse recipients and xenogeneically inhibit mouse T-cell proliferation and IFN-γ production in vitro. We recently established a new protocol for the isolation of a homogeneous population of MSCs called subfractionation culturing methods (SCM), and established a library of clonal MSC lines. Therefore, we also investigated whether MSCs isolated by the conventional gradient centrifugation method (GCM) and SCM show different efficacy in vivo. Intriguingly, clonal hMSCs (hcMSCs) isolated by SCM showed better efficacy than hMSCs isolated by GCM. Based on these results, the MHC-mismatched model of GvHD may be useful for evaluating the efficacy of human MSCs before the clinical trial. The results of this study suggest that different MSC lines may show different efficacy in vivo and in vitro.

Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells.

The objective of the study was to evaluate differentiation of human bone marrow mesenchymal stem cells into true or pseudo neurons after treating with chemical induction medium in vitro. The morphological changes were assessed using interference contrast microscopy. Immunocytochemistry and Western blotting were performed using neuronal markers. Further evaluation was conducted with proteomic profiling, DNA microarray analysis and the whole-cell patch clamp test. After three hours of treatment with chemical induction medium, nearly three-fourths of the hMSCs changed to cells with a neuronal phenotype. The results of immunocytochemistry and Western blotting showed a high expression of neuronal markers in these cells at 3 h which decreased at 24 h. The proteomics analysis showed no change of proteins related to neuronal differentiation. DNA microarray showed downregulation of neuron related genes. The patch clamp test was unable to demonstrate any similarity to true neurons. Our findings suggest that neuron-like cells derived from chemical induction of hMSCs are not the genuine neurons as they resemble true neurons phenotypically but are different in genotypic and electrophysiological characteristics.

The expression of hypoxia inducible factor-1alpha and apoptosis in herniated discs.

STUDY DESIGN: Human intervertebral disc was obtained for the study of hypoxia inducible factor (HIF)-1alpha and apoptosis using immunohistochemical staining. OBJECTIVE: To study the expression of HIF-1alpha and apoptosis in herniated lumbar discs. SUMMARY OF BACKGROUND DATA: The presence of HIF-1alpha in human chondrocytes and rat intervertebral discs has been proven; however, to our knowledge, its expression in human intervertebral disc cells has not been reported. Apoptosis of the human intervertebral disc appears as a degenerative change caused by the aging of the intervertebral disc. To our knowledge, there is no reported study showing the correlation between apoptosis and HIF-1alpha in the human intervertebral disc. METHODS: There were 15 human intervertebral discs stained for HIF-1alpha immunohistochemically, and apoptosis was detected using the terminal deoxynucleotidyl transferase mediated-dUTP nick end labeling method. On average, the patients were 32.9 years old. The intervertebral discs were divided into noncontained (9 patients) and contained (6) groups. For the control group, 5 disc samples were used. RESULTS: The expression of HIF-1alpha was visualized in every case, with an average of 62.2% +/- 9.5% in the noncontained group, 30.5% +/- 3.6% in the contained group, and 11.4% +/- 9.3% in the control group. Apoptosis occurred in 74.3% +/- 7.3% of the cells in the noncontained group, 42.8% +/- 5.5% of the cells in the contained group, and 28% +/- 8.4% of the cells in the control group. HIF-1alpha and apoptosis expressions were both observed more frequently in the noncontained disc herniation group (P < 0.001). The correlation analysis between the degree of HIF-1alpha expression and apoptosis was also statistically significant. CONCLUSIONS: HIF-1alpha and apoptosis physiologically occur in human beings. Their expression was the highest in the noncontained group. HIF-1alpha may play a crucial role for the survival of disc cells and resorption of the herniated disc in human intervertebral discs.

Posteromedial compartment cement extrusion after unicompartmental knee arthroplasty treated by arthroscopy: a case report.

Unicompartmental knee arthroplasty (UKA) with extrusion of cement into the posterior compartment of the knee is uncommon. Various problems after a UKA procedure, such as aseptic loosening, polyethylene wear and progressive arthritis have been reported. This study will report on a patient with extrusion of cement fragments into the posteromedial compartment of the knee after a UKA procedure. This complication was treated successfully with the direct posterior-posterior triangulation arthroscopic visualization method. In cementing the prosthesis, it is of paramount importance to take caution to completely remove extruded cement remnants in order to prevent this complication during UKA.