Adipogenic Induction Medium Research Articles

Cell-autonomous Action of the FGF19 Promotes UCP1 Expression and Function in Human Brown Adipocytes

Fibroblast growth factor 19 (FGF19) is an intestine-derived hormone that regulates glucose and energy metabolism. Central and systemic administration of human FGF19 in mice promotes increased energy expenditure and body weight loss through mechanisms that are not fully understood. In this study, we investigated whether FGF19 can activate thermogenesis in brown adipocytes (BA) in a cell-autonomous manner. We differentiated immortalized human brown preadipocytes into mature adipocytes with adipogenic induction media in the presence and absence of FGF19 for 18 days to evaluate the effects of FGF19 on adipogenesis and thermogenesis. Through qPCR and RNA-sequencing analyses, we observed that FGF19 receptors were expressed in both brown preadipocytes and mature BA. However, the obligatory FGF19 co-receptor, β-klotho, was not expressed in preadipocytes, suggesting that the effects of FGF19 must occur preferentially in mature BA. Lipid staining with Oil Red O as well as gene expression of the adipogenic markers fatty acid binding protein (FABP4) and peroxisome proliferator activated receptor-γ (PPAR-γ) showed that adipogenesis was not affected by FGF19. We then treated mature BA with FGF19 (100 ng/mL) acutely and chronically to evaluate FGF19-induced changes in signaling, gene transcription, and thermogenic function. FGF19 signaling was assessed by measuring ERK1/2 phosphorylation levels, which increased within 5 min and remained elevated for at least 30 min after treatment. Using Seahorse assays, we observed that 24-hour FGF19 treatment promoted increased basal respiration, ATP production, and proton leak in human BA, indictive of enhanced mitochondrial function. Since proton leak across the inner membrane through the uncoupling protein 1 (UCP1) generates heat, we assessed the expression of UCP1 and other thermogenic genes. Interestingly, FGF19 treatment specifically increased UCP1 gene expression without affecting other classical adrenergic-mediated thermogenic genes, such as deiodinase 2 (DIO2), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), PR domain containing 16 (PRDM16), and cell death-inducing DNA fragmentation factor A (CIDEA). These findings suggest that FGF19 regulates UCP1 expression through non-canonical pathways. Future work will elucidate the mechanism by which FGF19 promotes UCP1 gene expression in mature BA. Taken together, FGF19 stimulates ERK1/2 phosphorylation and UCP1 expression in human BA, leading to enhanced mitochondrial respiration. These results provide new mechanistic insights into FGF19’s effects on metabolic regulation. Topic subcategories: Adipocyte biology. Funding sources: Sao Paulo Research Foundation (FAPESP): 2022/15148-2 and NIH R01 DK102898. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Andrographis paniculata ameliorates estrogen deficiency-related osteoporosis by directing bone marrow mesenchymal stem cell fate.

Although Andrographis paniculata (AP) exhibits various biological functions such as anticancer, anti-inflammatory, antimalarial, antimicrobial, antioxidant, cardioprotective and immunomodulatory, its role in estrogen deficiency-related osteoporosis remains unclear. Ovariectomy (OVX)-induced estrogen deficiency-related osteoporotic mouse models and sham mouse models were established using 8-week-old female C57BL/6J mice. Micro-computed tomography (µCT) scanning was performed to assess the skeletal phenotype. The differentiation potential of bone marrow mesenchymal stem cells (BMSCs) from the OVX and sham groups was assessed by osteogenic or adipogenic induction medium in vitro. To verify the effects of AP, alizarin red S (ARS) staining, alkaline phosphatase (ALP) staining and oil red O (ORO) staining, reverse transcription assay and quantitative real-time polymerase chain reaction were applied to detect the lineage differentiation ability of BMSCs. µCT scanning showed that AP treatment attenuated the osteoporotic phenotype in OVX-induced estrogen deficiency-related osteoporotic mice. The results of ARS staining, ALP staining, ORO staining and quantitative real-time polymerase chain reaction indicated that BMSCs from OVX-induced osteoporotic mice displayed a significant reduction in osteogenic differentiation and an increase in adipogenic differentiation, which could be reversed by AP treatment. Our findings suggested that AP regulated the differentiation potential of BMSCs and ameliorated the development of estrogen deficiency-related osteoporosis, which might be an effective therapeutic method for estrogen deficiency-related osteoporosis.

In Vitro Evaluation of Human Demineralised Teeth Matrix on Osteogenic Differentiation of Gingival Mesenchymal Stem Cells

The use of tooth-derived material as a scaffold has gained attention recently due to its ease of availability and bioactive properties. Hence, the objective of this study was to determine in vitro interaction of human gingival mesenchymal stem cells (hGMSCs) with human demineralised teeth matrix (hDTM) on osteogenic potential with or without osteogenic inducers. The hGMSCs were established and characterised on their morphology, proliferation, population doubling time (PDT), viability, colony-forming ability, expression of cell surface markers and adipogenic differentiation. Further, the effect of hDTM on the biocompatibility and osteogenic differentiation ability of hGMSCs was evaluated. The hGMSCs displayed a fibroblast-like appearance and exhibited a greater proliferative activity. The cells showed > 91% viability, and PDT varied between 39.34 hours and 62.59 hours. Further, hGMSCs indicated their propensity to form clusters/ colonies, and expressed the markers, such as CD29, CD44, CD73 and CD90, but were negative for CD34 and CD45. When treated with adipogenic induction medium, hGMSCs were able to exhibit the formation of neutral lipid vacuoles. The hGMSCs cultured with hDTM did not show any cytotoxic changes including morphology and viability. Mineralisation of calcium nodules was observed in hGMSCs when cultured in osteogenic induction (OI) medium as an indication of osteogenesis. hGMSCs when cultured with hDTM confirmed the presence of a mineralised matrix. Further, when the cells were cultured with hDTM along with OI, they showed slightly enhanced differentiation into osteocytes. In conclusion, hGMSCs were shown to be biocompatible with hDTM, and demonstrated their enhanced osteogenic potential in the presence of hDTM and osteogenic supplements.

Isolation, culture and identification of SD rat bone marrow mesenchymal stem cells in tissue engineering

Seed cells are an important part of bone tissue engineering. In this study, we investigated methods for the isolation, culture and identification of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. BMSCs from SD rats were isolated and cultured using the whole bone marrow adherent method. Morphological observation and flow cytometry were used for the detection of cell surface markers. The osteogenic and adipogenic differentiation of cultured cells was induced and evaluated. BMSCs could be effectively isolated and purified using the whole bone marrow adherent method. The primary BMSCs cultured in vitro formed spindle-shaped, spiral and vigorous cell colonies. The flow cytometry results showed that CD90 was highly expressed on the cell surface, but CD34 and CD45 were not expressed. After culture in osteogenic and adipogenic induction medium, positive Alizarin Red and Oil Red O staining was observed, indicating the potential for multidirectional differentiation. The whole bone marrow adherent method is simple and practical, which makes the isolation, culture and amplification of BMSCs in vitro easy, and the method has little effect on cell activity and is an effective method for obtaining high-purity BMSCs.

Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis.

SummaryBone morphogenetic protein (BMP) cascades are upregulated during bone marrow-derived stromal cell (BMSC) chondrogenesis, contributing to hypertrophy and preventing effective BMSC-mediated cartilage repair. Previous work demonstrated that a proprietary BMP inhibitor prevented BMSC hypertrophy, yielding stable cartilage tissue. Because of the significant therapeutic potential of a molecule capable of hypertrophy blockade, we evaluated the capacity of a commercially available BMP type I receptor inhibitor with similar properties, LDN 193189, to prevent BMSC hypertrophy. Using 14-day microtissue chondrogenic induction cultures we found that LDN 193189 permitted BMSC chondrogenesis but did not prevent hypertrophy. LDN 193189 was sufficiently potent to counter mineralization and adipogenesis in response to exogenous BMP-2 in osteogenic induction cultures. LDN 193189 did not modify BMSC behavior in adipogenic induction cultures. Although LDN 193189 is effective in countering BMP signaling in a manner that influences BMSC fate, this blockade is insufficient to prevent hypertrophy.

CD44-positive cancer stem cells from oral squamous cell carcinoma exhibit reduced proliferation and stemness gene expression upon adipogenic induction.

We proposed to assess adipogenic differentiation and its effect on the proliferation and stemness markers in CD44 + OSCC CSCs. D44 + CSCs were sorted by magnetic sorting from the single-cell suspension of the OSCC tumor. Adipogenic differentiation was induced by an adipogenic induction medium. Lipid droplet formation was confirmed by oil red O staining. The expression of the cell surface marker was analyzed by flow cytometry. Real-time qPCR was performed to examine the gene expression activity. CD44 + OSCC CSCs can differentiate into adipocytes and adipogenesis in these cells decrease their proliferation and stemness gene expression. Adipogenic induction can make the cancer stem cells from OSCC tumors lose their stemness potential. Oral cancer, especially OSCC, is a huge burden worldwide. Similar to other stem cells, cancer stem cells can differentiate into other lineage cells. Our study shows that the proliferation and stemness gene expression in the CSCs from OSCC tumors can be thwarted by inducing them to differentiate into adipocytes, which could be advantageous to find out new clinical approaches in the treatment of cancers, like OSCC.

Characterization of porcine mesenchymal stromal cells and their proliferative and osteogenic potential in long-term culture

Background: Porcine mesenchymal stromal cells (pMSCs) are considered a valuable research model for bone tissue engineering, which requires adequate amounts of viable cells with sufficient potential for osteogenic differentiation. For isolation and expansion of these cells through long-term culture, appropriate culture conditions are needed. Objective: To study the effect of extended in vitro cultivation on pMSC proliferation and differentiation potential using different osteogenic and adipogenic induction media. Methods: pMSCs were isolated from the bone marrow of adult Göttingen minipigs, cultured, expanded to passage 20 (~160 days) and characterized by their expression of cell surface markers (wCD44, CD45, CD90, SWC9, fibronectin), alkaline phosphatase (ALP), and osteocalcin and their potential for osteogenic and adipogenic differentiation using different induction media. Results: pMSCs retained their capacity for proliferation and osteogenic differentiation, and the number of CD90-positive cells increased significantly over more than 60 population doublings. CD90 expression in uninduced cells correlated strongly with ALP expression following osteogenic induction. Medium enriched with calcium yielded a stronger osteogenic response. Conclusion: The selection of CD90-positive MSCs and adequate levels of calcium seem to enhance the osteogenic phenotype for bone tissue engineering.

Effect of volatile fatty acids on adipocyte differentiation in bovine dedifferentiated fat (DFAT) cells in vitro.

Here, we established dedifferentiated fat (DFAT) cells from mature bovine adipocytes and then examined the effects of volatile fatty acids on the differentiation of these DFAT cells into adipocytes in vitro. When mature adipocytes were isolated from bovine adipose tissue and cultured using the ceiling culture method, they were dedifferentiated into fibroblast-like cells without lipid droplets. These fibroblast-like cells, termed bovine DFAT (b-DFAT) cells, actively proliferated. After adipogenic induction, increased expression of adipocyte-specific genes occurred in b-DFAT cells and they redifferentiated into adipocytes with an accumulation of lipid droplets in their cytoplasm. The effects of volatile fatty acids on adipocyte differentiation in b-DFAT cells were also examined. Specifically, acetate, butyrate, and propionate added to adipogenic induction medium significantly enhanced the adipogenesis of b-DFAT cells compared with that observed in control cells; the addition of 10-3 mol of acetate enhanced adipogenesis of b-DFAT cells to the greatest extent. These results suggest that b-DFAT cells derived from bovine mature adipocytes are appropriate for the study of bovine adipocyte differentiation and that the optimum concentration treatment of acetate, a major energy source for ruminants, promotes adipogenesis of b-DFAT cells in vitro.

In vitro Comparison of Adipogenic Differentiation in Human Adipose-Derived Stem Cells Cultured with Collagen Gel and Platelet-Rich Fibrin.

Background: Adipose-derived stem cells (ADSCs) are the most preferred cell type, based on their phenotypic characteristics, plasticity, and favorable immunological properties for applications in soft-tissue augmentation. Hence, the present in vitro study was aimed to evaluate the adipogenic differentiation potential of human ADSCs upon culturing individually with collagen gel and platelet-rich fibrin (PRF). Materials and methods: The collected lipoaspirate was used for establishing ADSCs using enzymatic digestion method. Then, the cells were analyzed for their morphology, viability, proliferation rate, population doubling time (PDT), colony-forming ability, cell surface markers expression, and osteogenic differentiation as biological properties. Further, ADSCs were evaluated for their adipogenicity using induction media alone, and by culturing with collagen gel and PRF individually for prospective tissue augmentation. Results: ADSCs were successfully established in vitro and exhibited a fibroblast-like morphology throughout the culture period. Cells had higher viability, proliferation potential and showed their ability to form colonies. The positive expression of cell surface markers and osteogenic ability confirmed the potency of ADSCs. The ADSCs cultured on collagen gel and PRF, individually, showed higher number of differentiated adipocytes than ADSCs grown with adipogenic induction medium alone. Conclusion: The extent of lipid accumulation by ADSCs was slightly higher when cultured on collagen gel than on PRF. Additional experiments are required to confirm better suitability of scaffold materials for soft-tissue regeneration.

Inhibition of Notch Signaling Promotes the Differentiation of Epicardial Progenitor Cells into Adipocytes.

Background The role of Notch signaling pathway in the differentiation of epicardial progenitor cells (EPCs) into adipocytes is unclear. The objective is to investigate the effects of Notch signaling on the differentiation of EPCs into adipocytes. Methods Frozen sections of C57BL/6J mouse hearts were used to observe epicardial adipose tissue (EAT), and genetic lineage methods were used to trace EPCs. EPCs were cultured in adipogenic induction medium with Notch ligand jagged-1 or γ-secretase inhibitor DAPT. The adipocyte markers, Notch signaling, and adipogenesis transcription factors were determined. Results There was EAT located at the atrial–ventricular groove in mouse. By using genetic lineage tracing methods, we found that EPCs were a source of epicardial adipocytes. EPCs had lipid droplet accumulation, and the expression of adipocyte markers FABP-4 and perilipin-1 was upregulated under adipogenic induction. Activating the Notch signaling with jagged-1 attenuated the adipogenic differentiation of EPCs and downregulated the key adipogenesis transcription factor peroxisome proliferator activated receptor-γ (PPAR-γ), while inhibiting the signaling promoted adipogenic differentiation and upregulated PPAR-γ. When blocking PPAR-γ, the role of Notch signaling in promoting adipogenic differentiation was inhibited. Conclusions EPCs are a source of epicardial adipocytes. Downregulation of the Notch signaling pathway promotes the differentiation of EPCs into adipocytes via PPAR-γ.

Vitamin E and Selenium Facilitate the Osteogenesis and Adipogenesis of the Human Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells

Background and Aims: Previous studies have shown that adipose-derived mesenchymal stem/ stromal cells are one of the sources of mesenchymal stem cells (MSCs) with the capacity to differentiate into various mesodermal cell lineages. MSCs with cytokines secretion capability, which contributes to repair damaged tissues have gained wide credence for future cell-based therapeutic applications. In this study, the effect of the different dosages of vitamin E and Selenium was assessed on the stemness of the human adipose tissue-derived MSCs (AD-MSCs). Materials and Methods: Following 24 hours of cell treatments with different dosages of vitamin E and Selenium, MTT assay was used to assess the effect of them on cell proliferation. Moreover, the stemness of the AD-MSCs was assessed using osteogenic and adipogenic induction medium supplemented with the different dosages of the vitamin E and Selenium. Finally, Alizarin red and Oil-red O staining were performed to detect matrix mineralization and lipid droplet accumulation, respectively. Results: MTT data revealed that the optimal concentration for vitamin E and Selenium were 125 µM and 121 µM for the viability of the AD-MSCs. Moreover, the effect of vitamin E and Selenium were assessed by osteogenic and adipogenic differentiation by optimal dosages obtained by MTT assay, respectively. Maximum mineralization and lipid droplet aggregation of the differentiated cells were detected at IC50 in comparison with the control group. Conclusions: These results suggest that different dosages of vitamin E and Selenium could have various impacts on the proliferation and differentiation induction of human AD-MSCs.

Extracellular Vesicles Derived From Human Adipose-Derived Stem Cell Prevent the Formation of Hypertrophic Scar in a Rabbit Model.

BackgroundPreventing scar formation during wound healing has important clinical implications. Numerous studies have indicated that adipose-derived stem cell culture mediums, which are rich in cytokines and extracellular vesicles (EVs), regulate matrix remodeling and prevent scar formation after wound healing. Therefore, using a rabbit scar model, we tried to demonstrate which factor in adipose-derived stem cell culture mediums plays a major role in preventing scar formation (EVs or cytokines), as well as revealing the underlying mechanism.MethodsHuman adipose-derived stem cells (hASCs) were isolated from the subcutaneous adipose tissue of a healthy female donor. The surface CD markers of third-passage hASCs were analyzed by flow cytometry. The adipogenic differentiation capacity of the hASCs was detected using Oil O staining. A cultured medium of third- to five-passage hASCs was collected for EV and EV-free medium isolations. Extracellular vesicles were characterized using transmission electron microscopy, NanoSight, and the Western blotting for surface markers CD63, TSG101, and Alix. The EV-free medium was characterized by Western blotting for vascular endothelial growth factor A (VEGFA), platelet derived growth factor B (PDGFB), and transforming growth factor β 1 (TGFβ1). Eight-millimeter-diameter wounds were created on the ventral side of both ears of 16 New Zealand rabbits. A total of 0.1 mL of the human adipose-derived stem cell–extracellular vesicle (hASC-EV) or EV-free medium was locally injected into wounds made on the right ears during wound healing. Meanwhile, equal amounts of phosphate buffer saline were injected into the left ears as a control. Biopsies of the wounded skin and surrounding tissue were excised on postoperative day 28 and subjected to hematoxylin and eosin (H&E), Masson, and α-SMA immunofluorescence staining. The protein expression of α-SMA and collagen I in both scar tissues and the normal skin were evaluated via Western blotting.ResultsThe hASCs expressed high levels of 49d, CD90, CD105, and CD73 but did not express CD34 or CD45. The hASCs differentiated into adipocytes under an adipogenic induction medium. Under transmission electron microscopy, the hASC-EVs were circular, bilayer membrane vesicles and approximately 95% of the particles were between 50 and 200 nm in size. The hASC-EVs expressed the same surface markers as EVs, including CD63, TSG101, and Alix and displayed little expression of VEGFA, PDGFB, and TGFβ1. The EV-free medium had a high expression of VEGFA, PDGFB, and TGFβ1 but displayed no expression of CD63, TSG101, and Alix. In vivo, the hASC-EV treatment prevented the formation of hypertrophic scars on postoperative day 28 and suppressed collagen deposition and myofibroblast aggregation. However, the EV-free medium did not prevent the formation of hypertrophic scars on the same time point and had little effect on collagen deposition and myofibroblast aggregation when compared with the control group.ConclusionsOur study suggests that hASCs are associated with preventive scar formation therapy because of paracrine EVs rather than cytokines. A local injection of hASC-EVs during wound healing efficiently prevented hypertrophic scar formation, which may have a clinically beneficial antiscarring effect.

A Straightforward Approach to Engineer Vascularized Adipose Tissue Using Microvascular Fragments.

There is a need to overcome the donor-site morbidity and loss of volume over time that accompanies the current clinical approaches to treat soft tissue defects caused by disease and trauma. The development of bioactive constructs that can regenerate adipose tissue have made great progress toward addressing the limitations of current therapies, but their lack of vascularization and ability to meet the significant dimension requirements of tissue defects limit their clinical translatability. Microvascular fragments (MVFs) can form extensive vascular networks and contain resident cells that have the ability to differentiate into adipocytes. Therefore, the objective of this study was to determine if vascularized adipose tissue could be engineered using a fibrin-based hydrogel containing MVFs as the sole source of microvessels and adipocyte-forming cells. The potential for MVFs from different fat depots (epididymal, inguinal, and subcutaneous) to form microvascular networks and generate adipocytes when exposed to growth media (GM), adipogenic differentiation media (ADM), or when treated with GM before adipogenic induction (i.e., they were allowed to presprout before adipogenic induction) was evaluated. MVFs treated with adipogenic induction media, both with and without presprouting, contained lipid droplets, had an increase in expression levels of genes associated with adipogenesis (adiponectin and fatty acid synthase [FAS]), and had an increased rate of lipolysis. MVFs allowed to presprout before ADM treatment maintained their ability to form vascular networks while maintaining an elevated lipid content, adipogenic gene expression, and lipolysis rate. Collectively, these results support the contention that MVFs can serve as the sole source of biologic material for creating a vascularized adipose tissue scaffold. Impact statement Microvascular fragments have both the ability to form extensive vascular networks and function as a source of adipocytes. These phenomena were exploited as vascularized adipose tissue was generated by first allowing for a period of angiogenesis before the adipogenic induction. This strategy has the ability to provide a means of both improving soft tissue reconstruction while also serving as a model to better understand adipose tissue expansion.

Generation of Gellan Gum-Based Adipose-Like Microtissues.

Adipose tissue is involved in many physiological processes. Therefore, the need for adipose tissue-like analogues either for soft tissue reconstruction or as in vitro testing platforms is undeniable. In this work, we explored the natural features of gellan gum (GG) to recreate injectable stable adipose-like microtissues. GG hydrogel particles with different percentages of polymer (0.5%, 0.75%, 1.25%) were developed and the effect of obtained mechanical properties over the ability of hASCs to differentiate towards the adipogenic lineage was evaluated based on the expression of the early (PPARγ) and late (FABP4) adipogenic markers, and on lipids formation and accumulation. Constructs were cultured in adipogenic induction medium up to 21 days or for six days in induction plus nine days in maintenance media. Overall, no significant differences were observed in terms of hASCs adipogenic differentiation within the range of Young’s moduli between 2.7 and 12.9 kPa. The long-term (up to six weeks) stability of the developed constructs supported its application in soft tissue reconstruction. Moreover, their ability to function as adipose-like microtissue models for drug screening was demonstrated by confirming its sensitivity to TNFα and ROCK inhibitor, respectively involved in the repression and induction of the adipogenic differentiation.

Effects and mechanisms of up-regulation of heme oxygenase-1 on uric acid-induced adipocyte dysfunction

Objective To investigate the effects and mechanisms of up-regulation of heme oxygenase-1(HO-1)on uric acid-induced adipocyte dysfunction. Methods Human bone marrow-derived mesenchymal stem cells(MSCs)were cultured in vitro and second or third generation MSCs were selected and recultured in an adipogenic induction medium, with the addition of various amounts of fructose and uric acid to find the optimal concentrations.Then, the HO-1 inducer cobalt protoporphyrin(CoPP)and the HO-1 inhibitor tin porphyrin(SnMP)were added successively.There were five independent samples in each group.Adipogenesis in MSC-derived adipocytes and droplets were measured by spectrophotometry, expression levels of xanthine oxidase(XO)and NADPH were measured by Western blott, superoxide levels were measured by Luminous spectrometer, and levels of adipogenic markers and HO-1 were measured by reverse transcription-polymerase chain reaction(RT-PCR). Results Fructose at 500 μmol/L and uric acid at 50 mg/L were the optimal concentrations for stimulating adipogenesis in MSC-derived adipocytes(P<0.05). Compared with the controls, fructose significantly increased the levels of XO expression and uric acid(P<0.05), and concurrent treatment with fructose and CoPP effectively reversed both XO and uric acid levels to those of the controls(all P<0.05). However, SnMP negated the beneficial effects of CoPP(P<0.05). Additionally, uric acid decreased the number of small droplets and increased expression levels of adipogenic markers and NADPH(all P<0.05), but proadipogenic mediator levels were reduced with the addition of CoPP(all P<0.05). Furthermore, uric acid reduced expression levels of Wnt10b, but had no significant effect on HO-1 expression, andthese effects were reversed upon the addition of CoPP(all P<0.05). Conclusions Upregulation of HO-1 can reduce the levels of XO and uric acid, adipogenesis in MSC-derived adipocytes, and also the expression of adipogenic markers and NADPH.Therefore, it plays a role in antioxidant stress.HO-1 agonists may be targets for treating organ damage and controlling weight in elderly obese patients with metabolic syndrome. Key words: Heme oxygenase-1; Uric acid; Adipocytes; Oxidative stress

Adipogenic differentiation of human adipose derived mesenchymal stem cells in 3D architectured gelatin based hydrogels (ArcGel).

Polymeric matrices mimicking multiple functions of the ECM are expected to enable a material induced regeneration of tissues. Here, we investigated the adipogenic differentiation of human adipose derived mesenchymal stem cells (hADSCs) in a 3D architectured gelatin based hydrogel (ArcGel) prepared from gelatin and L-lysine diisocyanate ethyl ester (LDI) in an one-step process, in which the formation of an open porous morphology and the chemical network formation were integrated. The ArcGel was designed to support adipose tissue regeneration with its 3D porous structure, high cell biocompatibility, and mechanical properties compatible with human subcutaneous adipose tissue. The ArcGel could support initial cell adhesion and survival of hADSCs. Under static culture condition, the cells could migrate into the inner part of the scaffold with a depth of 840±120 μm after 4 days, and distributed in the whole scaffold (2 mm in thickness) within 14 days. The cells proliferated in the scaffold and the fold increase of cell number after 7 days of culture was 2.55±0.08. The apoptotic rate of hADSCs in the scaffold was similar to that of cells maintained on tissue culture plates. When cultured in adipogenic induction medium, the hADSCs in the scaffold differentiated into adipocytes with a high efficiency (93±1%). Conclusively, this gelatin based 3D scaffold presented high cell compatibility for hADSC cultivation and differentiation, which could serve as a potential implant material in clinical applications for adipose tissue reparation and regeneration.

A Rapid and Cost-Effective Protocol for Isolating Mesenchymal Stem Cells from the Human Amniotic Membrane

Background: Regarding the role of mesenchymal stem cells (MSCs) in regenerative medicine, many studies have been conducted to isolate these cells from various sources. In this study, a method was developed which will use only one enzyme and in the shortest time MSCs will be isolated from the amniotic membranes and expanded. Materials and Methods: The amniotic membrane (AM) was mechanically separated from the underlying membrane called chorionic. Then, the AM was sliced into tiny pieces and to isolate MSCs, it was digested only using collagens I instead of applying various enzymes. The isolated cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20% fetal bovine serum. The expression profiles of several markers at isolated cells were determined by flow cytometry. To assess the differentiation potential, the isolated cells were cultured in osteogenic and adipogenic induction media. Results: The results indicated that cells isolated from the AM expressed markers of CD44, CD105 and CD166 mesenchymal cells, but did not express CD34 and CD45 hematopoietic markers. The osteoblastic differentiation of the isolated cells was proven by alizarin red and alkaline phosphatase staining methods, whereas the adipogenic differentiation of the isolated cells was proven by Oil Red-O staining. Conclusion: The results of the study indicated that the isolated cells were of the MSCs family. Furthermore, it was demonstrated that MSCs can be obtained easily only by spending a short time and using one enzyme.[GMJ.2017;6(3):217-225] DOI: 10.22086/gmj.v0i0.670

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids.

Gingiva-derived stem cells have been applied for tissue-engineering purposes and may be considered a favorable source of mesenchymal stem cells as harvesting stem cells from the mandible or maxilla may be performed with ease under local anesthesia. The present study was performed to fabricate stem-cell spheroids using concave microwells and to evaluate the maintenance of stemness, viability, and differentiation potential. Gingiva-derived stem cells were isolated, and the stem cells of 4×105 (group A) or 8×105 (group B) cells were seeded into polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres and the change of the diameters of the spheroids were evaluated. The viability of spheroids was qualitatively analyzed via Live/Dead kit assay. A cell viability analysis was performed on days 1, 3, 6, and 12 with Cell Counting Kit-8. The maintenance of stemness was evaluated with immunocytochemical staining using SSEA-4, TRA-1-60(R) (positive markers), and SSEA-1 (negative marker). Osteogenic, adipogenic, and chondrogenic differentiation potential was evaluated by incubating spheroids in osteogenic, adipogenic and chondrogenic induction medium, respectively. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A than in group B. The majority of cells in the spheroids emitted green fluorescence, indicating the presence of live cells at day 6. At day 12, the majority of cells in the spheroids emitted green fluorescence, and a small portion of red fluorescence was also noted, which indicated the presence of dead cells. The spheroids were positive for the stem-cell markers SSEA-4 and TRA-1-60(R) and were negative for SSEA-1, suggesting that these spheroids primarily contained undifferentiated human stem cells. Osteogenic, adipogenic, and chondrogenic differentiation was more evident with an increase of incubation time: Mineralized extracellular deposits were observed following Alizarin Red S staining at days 14 and 21; oil globules were increased at day 18 when compared with day 6; and Alcian blue staining was more evident at day 18 when compared with day 6. Within the limits of this study, stem-cell spheroids from gingival cells maintained the stemness, viability, and differentiation potential during the experimental periods. This method may be applied for a promising strategy for stem-cell therapy.

Effects of Cimicifugae Rhizoma on the osteogenic and adipogenic differentiation of stem cells.

Cimicifugae Rhizoma, a herb with a long history of use in traditional Oriental medicine is reported to have anti-inflammatory, antioxidant, anti-complement and anticancer effects. The aim of the present study was to evaluate the effects of Cimicifugae Rhizoma extracts on the osteogenic and adipogenic differentiation of human stem cells derived from gingiva. Stem cells derived from gingiva were grown in the presence of Cimicifugae Rhizoma at final concentrations of 0.1, 1 and 10 µg/ml. Cell proliferation analyses were performed at day 15. For osteogenic differentiation experiments, the stem cells were cultured in osteogenic media containing β-glycerophosphate, ascorbic acid-2-phosphate and dexamethasone, and osteogenic differentiation was evaluated by analysis of osteocalcin expression at 21 days. For adipogenic differentiation experiments, the stem cells were grown in adipogenic induction medium, and the adipogenic differentiation was evaluated by analysis of adipocyte fatty acid-binding protein at day 14. The cultures grown in the presence of 0.1 µg/ml Cimicifugae Rhizoma showed a significant increase in cellular proliferation at day 15 compared with the control group. The relative osteogenic differentiation in the presence of Cimicifugae Rhizoma for the 0.1, 1 and 10 µg/ml groups was 171.5±13.7, 125.6±28.7 and 150.5±9.0, respectively, when that of the untreated control group on day 21 was considered to be 100%. The relative adipogenic differentiation at day 14 of the 0.1, 1 and 10 µg/ml groups in the presence of Cimicifugae Rhizoma was 97.5±15.0, 102.9±12.8 and 87.0±6.8%, respectively when that of the untreated control group on day 14 was considered to be 100%. Within the limits of this study, Cimicifugae Rhizoma increased the proliferation of stem cells derived from the gingiva, and low concentrations of Cimicifugae Rhizoma may increase the osteogenic differentiation of stem cells.

Vanadate Impedes Adipogenesis in Mesenchymal Stem Cells Derived from Different Depots within Bone.

Glucocorticoid-induced osteoporosis (GIO) is associated with an increase in bone marrow adiposity, which skews the differentiation of mesenchymal stem cell (MSC) progenitors away from osteoblastogenesis and toward adipogenesis. We have previously found that vanadate, a non-specific protein tyrosine phosphatase inhibitor, prevents GIO in rats, but it was unclear whether vanadate directly influenced adipogenesis in bone-derived MSCs. For the present study, we investigated the effect of vanadate on adipogenesis in primary rat MSCs derived from bone marrow (bmMSCs) and from the proximal end of the femur (pfMSCs). By passage 3 after isolation, both cell populations expressed the MSC cell surface markers CD90 and CD106, but not the hematopoietic marker CD45. However, although variable, expression of the fibroblast marker CD26 was higher in pfMSCs than in bmMSCs. Differentiation studies using osteogenic and adipogenic induction media (OM and AM, respectively) demonstrated that pfMSCs rapidly accumulated lipid droplets within 1 week of exposure to AM, while bmMSCs isolated from the same femur only formed lipid droplets after 3 weeks of AM treatment. Conversely, pfMSCs exposed to OM produced mineralized extracellular matrix (ECM) after 3 weeks, compared to 1 week for OM-treated bmMSCs. Vanadate (10 μM) added to AM resulted in a significant reduction in AM-induced intracellular lipid accumulation and expression of adipogenic gene markers (PPARγ2, aP2, adipsin) in both pfMSCs and bmMSCs. Pharmacological concentrations of glucocorticoids (1 μM) alone did not induce lipid accumulation in either bmMSCs or pfMSCs, but resulted in significant cell death in pfMSCs. Our findings demonstrate the existence of at least two fundamentally different MSC depots within the femur and highlights the presence of MSCs capable of rapid adipogenesis within the proximal femur, an area prone to osteoporotic fractures. In addition, our results suggest that the increased bone marrow adiposity observed in GIO may not be solely due to direct effect of glucocorticoids on bone-derived MSCs, and that an increase in femur lipid content may also arise from increased adipogenesis in MSCs residing outside of the bone marrow niche.