Adipose-derived Multipotent Stromal Cells Research Articles

Viable tendon neotissue from adult adipose-derived multipotent stromal cells.

Background: Tendon healing is frequently prolonged, unpredictable, and results in poor tissue quality. Neotissue formed by adult multipotent stromal cells has the potential to guide healthy tendon tissue formation. Objectives: The objective of this study was to characterize tendon neotissue generated by equine adult adipose-derived multipotent stromal cells (ASCs) on collagen type I (COLI) templates under 10% strain in a novel bioreactor. The tested hypothesis was that ASCs assume a tendon progenitor cell-like morphology, express tendon-related genes, and produce more organized extracellular matrix (ECM) in tenogenic versus stromal medium with perfusion and centrifugal fluid motion. Methods: Equine ASCs on COLI sponge cylinders were cultured in stromal or tenogenic medium within bioreactors during combined perfusion and centrifugal fluid motion for 7, 14, or 21days under 10% strain. Viable cell distribution and number, tendon-related gene expression, and micro- and ultra-structure were evaluated with calcein-AM/EthD-1 staining, resazurin reduction, RT-PCR, and light, transmission, and scanning electron microscopy. Fibromodulin was localized with immunohistochemistry. Cell number and gene expression were compared between culture media and among culture periods (p < 0.05). Results: Viable cells were distributed throughout constructs for up to 21days of culture, and cell numbers were higher in tenogenic medium. Individual cells had a round or rhomboid shape with scant ECM in stromal medium in contrast to clusters of parallel, elongated cells surrounded by highly organized ECM in tenogenic medium after 21days of culture. Transcription factor, extracellular matrix, and mature tendon gene expression profiles confirmed ASC differentiation to a tendon progenitor-like cell in tenogenic medium. Construct micro- and ultra-structure were consistent with tendon neotissue and fibromodulin was present in the ECM after culture in tenogenic medium. Conclusion: Long-term culture in custom bioreactors with combined perfusion and centrifugal tenogenic medium circulation supports differentiation of equine adult ASCs into tendon progenitor-like cells capable of neotissue formation.

Circulating osteoprogenitor (COP) cells have a mixed immune and mesenchymal progenitor function in humans.

Circulating osteoprogenitors (COP) are a population of cells in the peripheral circulation that possess functional and phenotypical characteristics of multipotent stromal cells (MSCs). This population has a solid potential to become an abundant, accessible, and replenishable source of MSCs with multiple potential clinical applications. However, a comprehensive functional characterization of COP cells is still required to test and fully develop their use in clinical settings. This study characterized COP cells by comparing them to bone marrow-derived MSCs (BM-MSCs) and adipose-derived MSCs (ASCs) through detailed transcriptomic and proteomic analyses. We demonstrate that COP cells have a distinct gene and protein expression pattern with a significantly stronger immune footprint, likely owing to their hematopoietic lineage. In addition, regarding progenitor cell differentiation and proliferation pathways, COP cells have a similar expression pattern to BM-MSCs and ASCs. COP cells are a unique but functionally similar population to BM-MSCs and ASCs, sharing their proliferation and differentiation capacity, thus presenting an accessible source of MSCs with strong potential for translational regenerative medicine strategies.

Feline Adipose Derived Multipotent Stromal Cell Transdifferentiation Into Functional Insulin Producing Cell Clusters.

Diabetes mellitus (DM) is one of the most prevalent feline endocrinopathies, affecting up to 1% of pet cats. De novo generation of functional insulin producing cell (IPC) clusters via transdifferentiation of feline adipose-derived multipotent stromal cells (ASCs) may not only provide a viable, functional cell therapy for feline DM, but may also serve as a platform for developing a comparable human treatment given feline and human DM similarities. Cells were induced to form IPCs with a novel, three-stage culture process with stromal or differentiation medium under static and dynamic conditions. Clusters were evaluated for intracellular zinc, viability, intracellular insulin, glucagon, and somatostatin, ultrastructure, glucose stimulated insulin secretion in the presence or absence of theophylline, and protein and gene expression. Isolated cells were multipotent, and cell clusters cultured in both media had robust cell viability. Those cultured in differentiation medium contained zinc and mono- or polyhormonal α-, β-, and δ-like cells based on immunohistochemical labeling and Mallory-Heidenhan Azan-Gomori’s staining. Ultrastructurally, cell clusters cultured in differentiation medium contained insulin granules within vesicles, and clusters had a concentration-dependent insulin response to glucose in the presence and absence of theophylline which increased both insulin secretion and intracellular content. Expression of NK6.1, Pax6, Isl1, Glut2, RAB3A, glucagon, insulin, and somatostatin increased with differentiation stage for both sexes, and expression of nestin at stages 1 and 2 and Neurod1 at stage 2 was higher in cells from female donors. The cluster insulin secretion responses and endocrine and oncogene gene expression profiles were inconsistent with insulinoma characteristics. A total of 180 proteins were upregulated in differentiated clusters, and the majority were associated with biological regulation, metabolic processes, or stimulus response. Dynamic culture of IPC clusters resulted in clusters composed of cells primarily expressing insulin that released higher insulin with glucose stimulation than those in static culture. Collectively, the results of this study support generation of functional IPC clusters using feline ASCs isolated from tissues removed during routine sterilization. Further, cluster functionality is enhanced with dynamic, motion-driven shear stress. This work establishes a foundation for development of strategies for IPC therapy for short or long-term diabetes treatment and may represent an option to study prevention and treatment of diabetes across species.

New approach to modification of poly (l-lactic acid) with nano-hydroxyapatite improving functionality of human adipose-derived stromal cells (hASCs) through increased viability and enhanced mitochondrial activity

The aim of this study was to determine the cytocompatibility of poly (l-lactide) (PLLA) scaffolds fabricated using co-rotating twin screw extrusion technique and functionalized with different concentrations of nano-hydroxyapatite (nHAp). The efforts were aimed on the designing bioactive scaffolds improving the viability and metabolic activity of human adipose-derived multipotent stromal cells (hASCs). The in vitro study was designed to determine the optimal nHAp concentration, based on analysis of hASCs morphology, adhesion rate, as well as metabolic and proliferative potential. Initially, the PLLA filled with three different concentrations of the nHAp were tested i.e. 5%, 10% and 15 wt%. The obtained results indicated that the 10 wt% nHAp in the PLLA (10% nHAp/PLLA) matrices improved the adhesion and proliferation of the hASCs, what was in good agreement with the results of tensile properties of the composites. Further, we performed profound studies regarding the cytotoxicity of 10% nHAp/PLLA. The analysis included the evaluation of the biomaterial influence on viability, apoptosis-related markers expression profile and mitochondrial function.The cytocompatibility of 10% nHAp/PLLA scaffolds toward the hASCs was confirmed. The hASCs propagated on 10% nHAp/PLLA were more viable then those propagated on the plain PLLA. The level of pro-apoptotic markers, i.e. caspase-3 and Bax in cultures on 10% nHAp/PLLA was significantly decreased. Obtained results correlated with higher mitochondrial membrane potential of hASCs in those cultures. The obtained composites may improve therapeutic potential of hASCs via directing their adhesion, enhancing proliferation and viability as well as increasing mitochondrial potential, thus may find potential application in tissue engineering.

Effects of enzyme and cryoprotectant concentrations on yield of equine adipose-derived multipotent stromal cells.

OBJECTIVE To evaluate effects of various concentrations of collagenase and dimethyl sulfoxide (DMSO) on yield of equine adipose-derived multipotent stromal cells (ASCs) before and after cryopreservation. SAMPLE Supragluteal subcutaneous adipose tissue from 7 Thoroughbreds. PROCEDURES Tissues were incubated with digests containing 0.1%, 0.05%, or 0.025% type I collagenase. Part of each resulting stromal vascular fraction was cryopreserved in 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12 and in 95% FBS and 5% DMSO. Half of each fresh and cryopreserved heterogeneous cell population was not immunophenotyped (unsorted) or was immunophenotyped for CD44+, CD105+, and major histocompatability complex class II (MHCII; CD44+-CD105+-MHCII+ cells and CD44+-CD105+-MHCII- cells). Cell proliferation (cell viability assay), plasticity (CFU frequency), and lineage-specific target gene and oncogene expression (reverse transcriptase PCR assays) were determined in passage 1 cells before and after culture in induction media. RESULTS Digestion with 0.1% collagenase yielded the highest number of nucleated cells. Cell surface marker expression and proliferation rate were not affected by collagenase concentration. Cryopreservation reduced cell expansion rate and CD44+-CD105+-MHCII- CFUs; it also reduced osteogenic plasticity of unsorted cells. However, effects appeared to be unrelated to DMSO concentrations. There were also variable effects on primordial gene expression among cell isolates. CONCLUSIONS AND CLINICAL RELEVANCE Results supported the use of 0.1% collagenase in an adipose tissue digest and 5% DMSO in cryopreservation medium for isolation and cryopreservation, respectively, of equine ASCs. These results may be used as guidelines for standardization of isolation and cryopreservation procedures for equine ASCs.

Collagen and Hydroxyapatite Scaffolds Activate Distinct Osteogenesis Signaling Pathways in Adult Adipose-Derived Multipotent Stromal Cells.

Osteogenic cell signaling pathway disruption varies among bone diseases. This investigation was designed to identify adipose-derived multipotent stromal cell (ASC) and bone graft scaffold combinations for local, targeted restoration of gene expression and extracellular matrix (ECM) deposition. Human ASC osteogenesis on bone graft materials was quantified following culture in stromal (S), osteogenic (O), or osteogenic for 48 h followed by stromal medium (OS) to test the two-part hypothesis: (1) identical ASC isolates on distinct bone graft scaffolds demonstrate unique viability, differentiation, ECM production, and gene expression in the same culture conditions; (2) identical ASC-bone graft scaffold combinations have different cell viability, differentiation, ECM production, and gene expression when cultured in S, O, or OS medium. Three commercially available bone graft scaffold materials, type I bovine collagen (C), hydroxyapatite + β-tricalcium phosphate + type I bovine collagen (HT), and β-tricalcium phosphate + type I bovine collagen (CT) were evaluated. Passage 3 ASCs were loaded onto scaffold blocks with a spinner flask bioreactor, and constructs were cultured up to 28 days. Cell viability, gene expression (alkaline phosphatase [ALPL], osteoprotegerin [TNFRSF11B], osteocalcin [BGLAP], cannabinoid receptors type I [CNR1] and II [CNR2], receptor activator of nuclear factor kappa β ligand [TNFSF11]), as well as ECM DNA, collagen, sulfated glycosaminoglycan, and protein content were quantified. Matrix organization was evaluated with scanning electron microscopy. Effects of scaffold, medium, or culture duration on cell viability were minimal. Significantly higher initial ALPL expression decreased with time, while BGLAP expression increased in HT constructs in O medium, and the constructs had the most abundant ECM components and ultrastructural organization. There was a similar, although delayed, pattern of gene expression and greater ECM collagen with less organization in C constructs in O medium. Higher CNR1 expression in C versus higher TNFRSF11B/TNFSF11 expression in HT constructs throughout the study support stimulation of unique osteogenic signaling pathways by identical cell isolates. These results suggest that bone scaffold composition may be used to selectively target specific osteogenic cell signaling pathways in ASC constructs to stimulate ECM deposition based on therapeutic needs.

Effects of Cryopreservation on Canine Multipotent Stromal Cells from Subcutaneous and Infrapatellar Adipose Tissue.

Adipose derived multipotent stromal cells (ASCs) isolated from brown versus white adipose tissues, may have distinct in vitro properties, including response to cryopreservation, due to differences in tissue physiology. This study was designed to determine the ultrastructure, immunophenotype, in vitro expansion capabilities and multipotentiality of paired canine ASCs harvested from subcutaneous (SUB) and infrapatellar (IFP) adipose tissue up to cell passage (P) 3 before and after cryopreservation. Adipocyte and ASC ultrastructure from the same tissue were distinct, and morphologies of both differed between tissue sources and with cryopreservation. Cell expansion and colony forming unit frequencies were similar between ASCs from both tissue sources before and after cryopreservation. Most fresh cells were CD29+, CD44+, CD90+ and CD34− up to P3. Cryopreserved P1 and P3 cells had lower percentages of CD29+ and 44+ cells, respectively, compared to fresh. Peroxisome proliferator-activated receptor γ (PPAR-γ) gene expression and sex determining region Y-box 2 (SOX2), CD29 and CD44 protein expression was lower in cryopreserved versus fresh P3 ASCs. Both PPAR-γ and osteopontin (OPN) protein expression increased in fresh and cryopreserved P3 ASCs cultured in adipogenic and osteogenic induction medium, respectively, while SOX2 decreased. Based on the study findings, in vitro expansion and multipotentiality are not distinct among canine SUB and IFP ASCs before or after cryopreservation. However, cryopreservation alters ASC ultrastructure, immunophenotype and transcription factor expression from both tissue sources. Future studies are necessary to determine the impact of cryopreservation on cell potential for therapy and de novo tissue generation.

The osteogenic properties of multipotent mesenchymal stromal cells in cultures on TiO₂ sol-gel-derived biomaterial.

The biocompatibility of the bone implants is a crucial factor determining the successful tissue regeneration. The aim of this work was to compare cellular behavior and osteogenic properties of rat adipose-derived multipotent stromal cells (ASCs) and bone marrow multipotent stromal cells (BMSCs) cultured on metallic substrate covered with TiO2 sol-gel-derived nanolayer. The morphology, proliferation rate, and osteogenic differentiation potential of both ASCs and BMSCs propagated on the biomaterials were examined. The potential for osteogenic differentiation of ASCs and BMSCs was determined based on the presence of specific markers of osteogenesis, that is, alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCL). Additionally, the concentration of calcium and phosphorus in extracellular matrix was determined using energy-dispersive X-ray spectroscopy (SEM-EDX). Obtained results showed that TiO2 layer influenced proliferation activity of ASCs, which manifested by shortening of population doubling time and increase of OPN secretion. However, characteristic features of cells morphology and growth pattern of cultures prompted us to conclude that ultrathin TiO2 layer might also enhance osteodifferentiation of BMSCs. Therefore in our opinion, both populations of MSCs should be used for biological evaluation of biomaterials compatibility, such results may enhance the area of investigations related to regenerative medicine.

Therapeutic doses of multipotent stromal cells from minimal adipose tissue.

Low yield of adult adipose-derived multipotent stromal cells (ASC) can limit autologous cell therapy in individuals with minimal adipose tissue. In this study, ASC isolation was optimized from approximately 0.2 g of feline epididymal adipose tissue for a treatment dose of 106–107 ASCs/kg. The ASC yield was determined for three digestions, 0.1 % collagenase in medium for 30 min (Classic), 0.3 % collagenase in buffer for 30 min (New) and 0.3 % collagenase in buffer for 1 h (Hour). After isolation by the new tissue digestion, continuously cultured ASCs (fresh) and cells recovered and expanded after cryostorage at P0 (revitalized) were characterized up to cell passage (P) 5. Outcomes included CD9, CD29, CD44, CD90 and CD105 expression, cell doublings and doubling times, fibroblastic, adipogenic and osteogenic colony forming unit (CFU) frequency percentages and lineage-specific target gene expression after induction. The New digestion had the highest CFU yield, and about 7x106 ASCs/kg were available within three cell passages (P2). Compared to earlier passages, target surface antigen expression was lowest in fresh P5 cells, and fresh and revitalized P3–5 cells had slower expansion. Fresh and revitalized P1 ASCs had higher CFU frequency percentages and lineage-specific gene expression than P3. The New method described in this study was most efficient for feline epididymal ASC isolation and did not alter in vitro cell behavior. Fresh and revitalized P0-P2 feline ASCs may be most effective for preclinical and clinical trials. This study offers a potential option for ASC isolation from limited adipose tissue resources across species.