Differentiation Of Adipose-derived Stromal/stem Cells Research Articles

BONE FROM FAT: ENHANCING OSTEOGENESIS FROM ADIPOSE STEM CELLS

Extensive bone defects, caused by severe trauma or resection of large bone tumors, are difficult to treat. Regenerative medicine, including stem cell transplantation, may provide a novel solution for these intractable problems and improve the quality of life in affected patients. Adipose-derived stromal/stem cells (ASCs) have been extensively studied as cell sources for regenerative medicine due to their excellent proliferative capacity and the ability to obtain a large number of cells with minimal donor morbidity. However, the osteogenic potential of ASCs is lower than that of bone marrow-derived stromal/stem cells. To address this disadvantage, our group has employed various methods to enhance osteogenic differentiation of ASCs, including factors such as bone morphogenetic protein or Vitamin D, coculture with bone marrow stem cells, VEGF transfection, and gene transfer of Runx-2 and osterix. Recently, we mined a marker that can predict the osteogenic potential of ASC clones and also investigated the usefulness of the molecule as the enhancer of osteogenic differentiation of ASCs as well as its mechanism of action. Through RNA-seq gene analysis, we discovered that GSTT1 was the most distinguished gene marker between highly osteogenic and poorly osteogenic ASC clones. Knockdown of GSTT1 in high osteogenic ASCs by siGSTT1 treatment reduced mineralized matrix formation while GSTT1 overexpression by GSTT1 transfection or GSTT1 recombinant protein treatment enhanced osteogenic differentiation of low osteogenic ASCs. Metabolomic analysis confirmed significant changes of metabolites related to bone differentiation in ASCs transfected with GSTT1. A high total antioxidant capacity, low levels of cellular reactive oxygen species and increased GSH/GSSG ratios were also detected in GSTT1- transfected ASCs. GSTT1 can be a useful marker to screen the highly osteogenic ASC clones and also a therapeutic factor to enhance the osteogenic differentiation of poorly osteogenic ASC clones.

Combination of Adipose-Derived Stromal/Stem Cells and Decellularized Adipose Tissue Hydrogel for Osteogenic Applications.

Adipose-derived stromal/stem cells (ASCs) and decellularized adipose tissue (DAT) are adipose tissue products obtained from individuals undergoing fat removal procedures like liposuction, lipectomy, or breast reduction. DAT hydrogel is prepared by removing the cells from the adipose tissue and digesting it to form a liquid material that forms a gel at physiological temperature. ASCs seeded on DAT have displayed osteogenic potential in vitro and in animal models of bone defects. Herein, we describe the methods for preparing DAT hydrogel, ASC seeding in DAT hydrogel, osteogenic differentiation of ASCs, creation of critical-sized femur defect model in mice, its treatment with ASC-DAT hydrogel, and analyses.

GSTT1 as a Predictive Marker and Enhancer for Osteogenic Potential of Human Adipose-Derived Stromal/Stem Cells.

Adipose-derived stromal/stem cells (ASCs) have been extensively studied as cell sources for regenerative medicine for bone because of their excellent proliferative capacity and the ability to obtain a large number of cells with minimal donor morbidity. On the other hand, the differentiation potential of ASCs is generally lower than that of bone marrow-derived stromal/stem cells and varies greatly depending on donors. In this study, we mined a marker that can predict the osteogenic potential of ASC clones and also investigated the usefulness of the molecule as the enhancer of osteogenic differentiation of ASCs as well as its mechanism of action. Through RNA-seq gene analysis, we discovered that GSTT1 (Glutathione S-transferase theta-1) was the most distinguished gene marker between highly osteogenic and poorly osteogenic ASC clones. Knockdown of GSTT1 in high osteogenic ASCs by siGSTT1 treatment reduced mineralized matrix formation. On the other hand, GSTT1 overexpression by GSTT1 transfection or GSTT1 recombinant protein treatment enhanced osteogenic differentiation of low osteogenic ASCs. Metabolomic analysis confirmed significant changes of metabolites related to bone differentiation in ASCs transfected with GSTT1. A high total antioxidant capacity, low levels of cellular reactive oxygen species, and increased GSH/GSSG ratios were also detected in GSTT1-transfected ASCs. When the in vivo effect of GSTT1-transfected ASCs on bone regeneration was investigated with segmental long-bone defect model in rats, bone regeneration was significantly better after implantation of GSTT1-transfected ASCs compared with that of control vector-transfected ASCs. In conclusion, GSTT1 can be a useful marker to screen the highly osteogenic ASC clones and also a therapeutic factor to enhance the osteogenic differentiation of poorly osteogenic ASC clones. © 2023 American Society for Bone and Mineral Research (ASBMR).

Human adipose tissue-derived small extracellular vesicles promote soft tissue repair through modulating M1-to-M2 polarization of macrophages

BackgroundSuccessful regenerative medicine strategies need the manipulation and control of macrophages’ phenotypic switching. Our previous study indicated that rat and porcine adipose tissue-derived small extracellular vesicles could successfully promote soft tissue repair. However, whether human adipose tissue-derived small extracellular vesicles (h-sEV-AT) showed the same ability to promote soft tissue regeneration and whether adipose tissue-derived small extracellular vesicles (sEV-AT) contribute to modulating the polarization of macrophages were unknown.MethodsIn this study, we, for the first time, isolated h-sEV-AT from liposuction adipose tissue and characterized the morphology, size distribution, and marker protein. In vitro, we treated adipose-derived stromal/stem cells (ASCs), endothelial cells (ECs), and M1 macrophages with h-sEV-AT. In vivo, the ability of h-sEV-AT to promote soft tissue regeneration and polarize macrophages was investigated.ResultsThe results indicated that h-sEV-AT possessed the characteristics of small extracellular vesicles (sEVs). In vitro, an obvious increase in adipogenesis and angiogenesis was induced by h-sEV-AT. In vivo, h-sEV-AT successfully induced the regeneration of adipose tissue and effectively accelerated full-thickness skin wound healing. Besides, we found that h-sEV-AT showed the ability to increase the percentage of M2 macrophages both in vivo and in vitro, which had been reported to contribute to tissue repair and regeneration.ConclusionsTaken together, these results suggested that h-sEV-AT showed the ability to induce soft tissue repair supported by not only the differentiation of ASCs and ECs but also the polarization of macrophages. Considering the abundant sources, high yield, and guaranteed effectiveness, this study provided a cell-free strategy for soft tissue regeneration that directly isolated small extracellular vesicles from human liposuction adipose tissue.

Human Adipose-Derived Hydrogel Characterization Based on In Vitro ASC Biocompatibility and Differentiation.

Hydrogels serve as three-dimensional scaffolds whose composition can be customized to allow attachment and proliferation of several different cell types. Extracellular matrix-derived hydrogels are considered close replicates of the tissue microenvironment. They can serve as scaffolds for in vitro tissue engineering and are a useful tool to study cell-scaffold interaction. The aim of the present study was to analyze the effect of adipose-derived stromal/stem cells (ASCs) and decellularized adipose tissue-derived (DAT) hydrogel interaction on ASC morphology, proliferation, differentiation, and DAT hydrogel microstructure. First, the ASCs were characterized using flow cytometry, adipogenic/osteogenic differentiation, colony-forming unit fibroblast assay and doubling time. The viability and proliferation assays showed that ASCs seeded in DAT hydrogel at different concentrations and cultured for 21 days remained viable and displayed proliferation. ASCs were seeded on DAT hydrogel and cultured in stromal, adipogenic, or osteogenic media for 14 or 28 days. The analysis of adipogenic differentiation demonstrated the upregulation of adipogenic marker genes and accumulation of oil droplets in the cells. Osteogenic differentiation demonstrated the upregulation of osteogenic marker genes and mineral deposition in the DAT hydrogel. The analysis of DAT hydrogel fiber metrics revealed that ASC seeding, and differentiation altered both the diameter and arrangement of fibers in the matrix. Matrix metalloproteinase-2 (MMP-2) activity was assessed to determine the possible mechanism for DAT hydrogel remodeling. MMP-2 activity was observed in all ASC seeded samples, with the osteogenic samples displaying the highest MMP-2 activity. These findings indicate that DAT hydrogel is a cytocompatible scaffold that supports the adipogenic and osteogenic differentiation of ASCs. Furthermore, the attachment of ASCs and differentiation along adipogenic and osteogenic lineages remodels the microstructure of DAT hydrogel.

Increase of gremlin 2 with age in human adipose-derived stromal/stem cells and its inhibitory effect on adipogenesis.

IntroductionAdipose-derived stromal/stem cells (ASCs) have attracted attention as a promising material for regenerative medicine. Previously, we reported an age-related decrease in the adipogenic potential of ASCs from human subjects and found that the individual difference in this potential increased with age, although the mechanisms remain unclear. Recently, other groups demonstrated that a secreted antagonist of bone morphogenetic protein (BMP) signaling, Gremlin 2 (GREM2), inhibits the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into osteoblasts and the adipogenesis of 3T3-L1 cell. Here, we examined the effects of GREM2 on the differentiation of ASCs into adipocytes.MethodsTo examine changes in GREM2 expression levels with age, immunohistochemistry was performed on subcutaneous adipose tissues from subjects 12–97 years of age. Next, GREM2 gene expression levels in ASCs collected from subjects 5–90 years of age were examined by RT-PCR, and the change with age and correlation between the expression level and the adipogenic potential of ASCs were analyzed. In addition, to assess whether GREM2 affects adipogenesis, ASCs (purchased from a vendor) were cultured to induce adipogenesis with recombinant GREM2 protein, and siRNA-induced GREM2 knockdown experiment was also performed using aged ASCs.ResultsIn adipose tissues, GREM2 expression was observed in cells, including ASCs, but not in mature adipocytes, and the expression level per cell increased with age. GREM2 expression levels in ASCs cultured in vitro also increased with age, and the individual differences in the level increased with age. Of note, partial correlation analysis controlled for age revealed that the adipogenic potential of ASCs and the GREM2 gene expression level were negatively correlated. Furthermore, based on a GREM2 addition experiment, GREM2 has inhibitory effects on the adipogenesis of ASCs through activation of Wnt/β-catenin signaling. On the other hand, GREM2 knockdown in aged ASCs promoted adipogenesis.ConclusionsThe GREM2 expression level was confirmed to play a role in the age-related decrease in adipogenic potential observed in ASCs isolated from adipose tissues as well as in the enhancement of the individual difference, which increased with age. GREM2 in adipose tissues increased with age, which suggested that GREM2 functions as an inhibitory factor of adipogenesis in ASCs.

Transcriptomic Profiling of Adipose Derived Stem Cells Undergoing Osteogenesis by RNA-Seq

Adipose-derived stromal/stem cells (ASCs) are multipotent in nature that can be differentiated into various cells lineages such as adipogenic, osteogenic, and chondrogenic. The commitment of a cell to differentiate into a particular lineage is regulated by the interplay between various intracellular pathways and their resultant secretome. Similarly, the interactions of cells with the extracellular matrix (ECM) and the ECM bound growth factors instigate several signal transducing events that ultimately determine ASC differentiation. In this study, RNA-sequencing (RNA-Seq) was performed to identify the transcriptome profile of osteogenic induced ASCs to understand the associated genotype changes. Gene ontology (GO) functional annotations analysis using Database for Annotation Visualization and Integrated Discovery (DAVID) bioinformatics resources on the differentially expressed genes demonstrated the enrichment of pathways mainly associated with ECM organization and angiogenesis. We, therefore, studied the expression of genes coding for matrisome proteins (glycoproteins, collagens, proteoglycans, ECM-affiliated, regulators, and secreted factors) and ECM remodeling enzymes (MMPs, integrins, ADAMTSs) and the expression of angiogenic markers during the osteogenesis of ASCs. The upregulation of several pro-angiogenic ELR+ chemokines and other angiogenic inducers during osteogenesis indicates the potential role of the secretome from differentiating ASCs in the vascular development and its integration with the bone tissue. Furthermore, the increased expression of regulatory genes such as CTNNB1, TGBR2, JUN, FOS, GLI3, and MAPK3 involved in the WNT, TGF-β, JNK, HedgeHog and ERK1/2 pathways suggests the regulation of osteogenesis through interplay between these pathways. The RNA-Seq data was also validated by performing QPCR on selected up- and down-regulated genes (COL10A1, COL11A1, FBLN, FERMT1, FN1, FOXF1, LAMA3, LAMA4, LAMB1, IGF1, WNT10B, MMP1, MMP3, MMP16, ADAMTS6, and ADAMTS14).

Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation

ABSTRACTStromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

Gap junctional intercellular communication in adipose-derived stromal/stem cells is cell density-dependent and positively impacts adipogenic differentiation.

Adipose-derived stromal/stem cells (ASCs) represent a widely used cell source with multi-lineage differentiation capacity in approaches for tissue engineering and regenerative medicine. Despite the multitude of literature on their differentiation capacity, little is reported about the physiological properties contributing to and controlling the process of lineage differentiation. Direct intercellular communication between adjacent cells via gap junctions has been shown to modulate differentiation processes in other cell types, with connexin 43 (Cx43) being the most abundant isoform of the gap junction-forming connexins. Thus, in the present study we focused on the expression of Cx43 and gap junctional intercellular communication (GJIC) in human ASCs, and its significance for adipogenic differentiation of these cells. Cx43 expression in ASCs was demonstrated histologically and on the gene and protein expression level, and was shown to be greatly positively influenced by cell seeding density. Functionality of gap junctions was proven by dye transfer analysis in growth medium. Adipogenic differentiation of ASCs was shown to be also distinctly elevated at higher cell seeding densities. Inhibition of GJIC by 18α-glycyrrhetinic acid (AGA) significantly compromised adipogenic differentiation, as demonstrated by histology, triglyceride quantification, and adipogenic marker gene expression. Flow cytometry analysis showed a lower proportion of cells undergoing adipogenesis when GJIC was inhibited, further indicating the importance of GJIC in the differentiation process. Altogether, this study demonstrates the impact of direct cell-cell communication via gap junctions on the adipogenic differentiation process of ASCs, and may contribute to further integrate direct intercellular crosstalk in rationales for tissue engineering approaches.

Standardization of platelet releasate products for clinical applications in cell therapy: a mathematical approach

BackgroundStandardized animal-free components are required for manufacturing cell-based medicinal products. Human platelet concentrates are sources of growth factors for cell expansion but such products are characterized by undesired variability. Pooling together single-donor products improves consistency, but the minimal pool sample size was never determined.MethodsSupernatant rich in growth factors (SRGF) derived from n = 44 single-donor platelet-apheresis was obtained by CaCl2 addition. n = 10 growth factor concentrations were measured. The data matrix was analyzed by a novel statistical algorithm programmed to create 500 groups of random data from single-donor SRGF and to repeat this task increasing group statistical sample size from n = 2 to n = 20. Thereafter, in created groups (n = 9500), the software calculated means for each growth factor and, matching groups with the same sample size, the software retrieved the percent coefficient of variation (CV) between calculated means. A 20% CV was defined as threshold. For validation, we assessed the CV of concentrations measured in n = 10 pools manufactured according to algorithm results. Finally, we compared growth rate and differentiation potential of adipose-derived stromal/stem cells (ASC) expanded by separate SRGF pools.ResultsGrowth factor concentrations in single-donor SRGF were characterized by high variability (mean (pg/ml)–CV); VEGF: 950–81.4; FGF-b: 27–74.6; PDGF-AA: 7883–28.8; PDGF-AB: 107834–32.5; PDGF-BB: 11142–48.4; Endostatin: 305034–16.2; Angiostatin: 197284–32.9; TGF-β1: 68382–53.7; IGF-I: 70876–38.3; EGF: 2411–30.2). In silico performed analysis suggested that pooling n = 16 single-donor SRGF reduced CV below 20%. Concentrations measured in 10 pools of n = 16 single SRGF were not different from mean values measured in single SRGF, but the CV was reduced to or below the threshold. Separate SRGF pools failed to differently affect ASC growth rate (slope pool A = 0.6; R2 = 0.99; slope pool B = 0.7; R2 0.99) or differentiation potential.DiscussionResults deriving from our algorithm and from validation utilizing real SRGF pools demonstrated that pooling n = 16 single-donor SRGF products can ameliorate variability of final growth factor concentrations. Different pools of n = 16 single donor SRGF displayed consitent capability to modulate growth and differentiation potential of expanded ASC. Increasing the pool size should not further improve product composition.

Comparison of Stromal/Stem Cells Isolated from Human Omental and Subcutaneous Adipose Depots: Differentiation and Immunophenotypic Characterization

The emerging field of regenerative medicine has identified adipose tissue as an abundant source of stromal/stem cells for tissue engineering applications. Therefore, we have compared the differentiation and immunophenotypic features of adipose-derived stromal/stem cells (ASC) isolated from either omental or subcutaneous adipose depots. Human tissue samples were obtained from bariatric and plastic surgical practices at a university-affiliated teaching hospital and a private practice, respectively, with informed patient consent. Primary cultures of human ASC were isolated from adipose specimens within 24 h of surgery and culture expanded in vitro. The passaged ASC were induced to undergo adipogenic or osteogenic differentiation as assessed by histochemical methods or evaluated for surface antigen expression profiles by flow cytometry. ASC yields per unit weight of tissue were comparable between omental and subcutaneous depots. At passage 0, the immunophenotype of omental and subcutaneous ASC were not significantly different with the exception of CD105 and endoglin, a component of the transforming growth factor β receptor. The adipogenic differentiation of omental ASC was less robust than that of subcutaneous ASC based on in vitro histochemical and PCR assays. Although the yield and immunophenotype of ASC from omental adipose depots resembled that of subcutaneous ASC, omental ASC displayed significantly reduced adipogenic differentiation capacity following chemical induction. Further studies are necessary to evaluate and optimize the differentiation function of omental ASC in vitro and in vivo. Pending such analyses, omental ASC should not be used interchangeably with subcutaneous ASC for regenerative medical applications.

Analysis of the Pro- and Anti-Inflammatory Cytokines Secreted by Adult Stem Cells during Differentiation.

Adipose-derived stromal/stem cells (ASCs) are adult stem cells that have the potential to differentiate into mesenchymal lineage cells. The abundance of ASCs in adipose tissue and easy accessibility with relatively little donor site morbidity make them attractive candidate cells for tissue engineering and regenerative medicine. However, the underlying inflammatory process that occurs during ASC differentiation into adipocytes and osteoblast has not been extensively investigated. ASCs cultured in osteogenic and adipogenic differentiation medium were characterized by oil red o staining and alizarin red staining, respectively. ASCs undergoing osteogenic and adipogenic differentiation were isolated on days 7, 14, and 21 and assessed by qRT-PCR for the expression of pro- and anti-inflammatory cytokines. ASCs undergoing osteogenic differentiation expressed a distinct panel of cytokines that differed from the cytokine profile of ASCs undergoing adipogenic differentiation at each of the time points analyzed. Mapping the cytokine expression profile during ASC differentiation will provide insight into the role of inflammation in this process and identify potential targets that may aid in enhancing osteogenic or adipogenic differentiation for the purposes of tissue engineering and regenerative medicine.

During epithelial differentiation of human adipose-derived stromal/stem cells, expression of zonula occludens protein-1 is induced by a combination of retinoic acid, activin-A and bone morphogenetic protein-7

Adipose-derived stromal/stem cells (ASC) possess a multilineage differentiation potential, can be used from an autologous origin, and are, therefore, attractive candidates for clinical applications to repair or regenerate damaged tissues and organs. Beside their well-known differentiation into cells of mesodermal origin, ASC are able to differentiate into cells of ecto- and endodermal origin. Previous studies have shown that all trans retinoic acid (ATRA) induces the expression of cytokeratin 18 (CK18), indicating the beginning of differentiation into the epithelial lineage. Nevertheless, ATRA does not induce the expression of other epithelial markers. Therefore, we tested the additional influence of two growth factors on the onset of epithelial differentiation of ASC. The cells were cultured with ATRA, Activin A (ActA) and bone morphogenetic protein-7 (BMP-7), either alone or in combination. Differentiation into the epithelial lineage was assessed by the expression of the characteristic epithelial markers CK18 and zonula occludens protein 1 (ZO-1) using Western blot, immunofluorescence staining and polymerase chain reaction (PCR) analysis. The mixture of all three factors induced epithelial differentiation of ASC without enhancing cell proliferation. Upon induction, the ASC showed phenotypic changes consistent with an epithelial phenotype. The addition of the growth factors ActA and BMP-7 enhanced the inductive effect of ATRA, as shown by the de novo expression of ZO-1 in addition to CK18 expression. Our study highlights the onset of the epithelial differentiation of ASC induced by culture with a combination of ATRA, ActA and BMP-7.

The Effect of Storage Time on Adipose-Derived Stem Cell Recovery from Human Lipoaspirates

Multipotent adipose-derived stromal/stem cells (ASCs) can be isolated with high yield from human subcutaneous lipoaspirates. This study reports our experience isolating, expanding, differentiating and immunophenotypically characterizing ASCs over a period of 4 days after having surgically obtained the lipoaspirate samples. The ultimate goal is to understand how to optimize the consistent isolation of ASCs from lipoaspirates. The length of time between adipose tissue harvest and processing will need to be systematically evaluated with respect to cell yield, viability, and function since some distance is likely to exist between the plastic surgeon’s office where lipoaspiration is performed and the current Good Manufacturing Practices (cGMP) laboratory where the ASCs are isolated. The objective of this study was to determine the effect of time delays on the yield and function of ASCs after collagenase digestion. We were able to isolate ASCs from lipoaspirates up to 72 h after the surgical procedure. The ASCs isolated on sequential days after the original tissue harvest proliferated, differentiated and maintained cell surface markers. We found that the initial 24-hour period is optimal for isolating ASCs with respect to cell yield and that there was no significant difference between ASC cell proliferation and differentiation ability within this period of time. In contrast, each of these parameters declined significantly for tissues maintained at room temperature for 48 or 72 h prior to isolation. These findings should be considered in the future development of standard operating procedures for cGMP processing of clinical-grade human ASCs.

Adipose tissue as a stem cell source for musculoskeletal regeneration

Adipose tissue is an abundant, easily accessible, and reproducible cell source for musculo-skeletal regenerative medicine applications. Initial derivation steps yield a heterogeneous population of cells of stromal vascular fraction (SVF) cells. Subsequent adherent selection of the SVF results in a relatively homogeneous population of adipose-derived stromal/stem cells (ASCs) capable of adipogenic, chondrogenic, myogenic, and osteogenic differentiation in vitro on scaffolds in bioreactors and in vivo in pre-clinical animal models. Unlike hematopoietic cells, ASCs do not elicit a robust lymphocyte reaction and instead release immunosuppressive factors, such as prostaglandin E2. These immunomodulatory features suggest that allogeneic and autologous ASCs will engraft successfully for tissue regeneration purposes. The differentiation and expansion potential of ASCs can be modified by growth factors, bio-inductive scaffolds, and bioreactors providing environmental control and biophysical stimulation. Gene therapy approaches using lentiviral transduction can be used to direct differentiation of ASCs to particular lineages. We discuss the utility of ASCs for musculo-skeletal tissue repair and some of the technologies that can be implemented to unlock the full regenerative potential of these highly valuable cells.