Effects of melatonin on the proliferation and differentiation of rat adipose-derived stem cells

ABSTRACT Background: Osteogenesis driven by adipose-derived stem cells (ADSCs) is regulated by physiological and pathological factors. Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may have an influence on bone formation. However, little is known about the effects of melatonin on osteogenesis, which thus remains to be elucidated. This study was performed to determine whether melatonin at physiological concentrations (0.01-10 nM) could affect the in vitro proliferation and osteogenic differentiation of rat ADSCs.Materials and Methods: ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced in a monolayer culture using osteogenic medium with or without melatonin at physiological concentrations (0.01-10 nM). After four weeks, the cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP) activity using an ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and flow cytometry, respectively.Results: The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to osteogenic medium alone. Similarly, mineral deposition (calcium level) also decreased in the presence of melatonin. Flow cytometry confirmed that cell growth had decreased and that the numbers of apoptotic cells had increased.Conclusion: These results suggest that the physiological concentration of melatonin has a negative effect on ADSC osteogenesis.

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188 The Effect of Vitamin B12 on the Differentiation of Adipose-Derived Stem Cells into Osteoblasts

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab188 ◽

2018 ◽

Vol 30 (1) ◽

pp. 234

Author(s):

T. A. Bane ◽

J. C. Bertels ◽

K. M. Polkoff ◽

M. Rubessa ◽

M. B. Wheeler

Keyword(s):

Stem Cells ◽

Vitamin B12 ◽

Difficult Problem ◽

Negative Control ◽

Nodule Formation ◽

Adipose Derived Stem Cells ◽

Osteogenic Medium ◽

Alizarin Red ◽

Α Level

Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules offer a potential solution to this difficult problem. The aim of this project was to evaluate if the osteogenic medium infused with vitamin B12 influences the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Vitamin B12 has been shown to have a stimulatory effect on osteoclastogenesis in vitro (Vaes et al. 2009 Calcified Tissue Int. 84, 413-422). Our hypothesis was that the presence of vitamin B12 in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). The ASC were divided in 8 different treatments: 8 concentrations of vitamin B12 in the osteogenic medium (0.1, 0.2, 1, 2, 10, and 20 μM) plus 2 control treatments (osteogenic medium without vitamin B12 and a negative control, DMEM). The medium was changed twice a week for 4 weeks. The experiment was replicated 6 times. At the end of the culture period, cells were stained with Alizarin Red and Von Kossa stains. In each well, we counted the nodules and then divided them in 2 categories: formed and forming nodules. Data was analysed using the generalized linear model (GLM) procedure in SPSS (IBM/SPSS, Armonk, NY, USA). Bonferroni’s post hoc test was used to perform statistical multiple comparison. The α-level was set at 0.01. The results showed that the concentration of 20 μM vitamin B12 was detrimental for nodule formation. Table 1 illustrates the number of formed and forming nodules in addition to their standard deviation. There was no positive effect on nodule formation when different concentrations of vitamin B12 were added to the osteogenic medium. More experiments need to be conducted to determine if vitamin B12 can act synergistically with other vitamins to produce a significant role in ASC differentiation into osteoblasts. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with vitamin B12 incorporated into their matrix. Table 1.The average number of formed and forming osteoblast nodules compared between treatment groups (SD in parentheses)

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Comparing the Osteogenic Potential and Bone Regeneration Capacities of Dedifferentiated Fat Cells and Adipose-Derived Stem Cells In Vitro and In Vivo: Application of DFAT Cells Isolated by a Mesh Method

International Journal of Molecular Sciences ◽

10.3390/ijms222212392 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12392

Author(s):

Kiyofumi Takabatake ◽

Masakazu Matsubara ◽

Eiki Yamachika ◽

Yuki Fujita ◽

Yuki Arimura ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Potential ◽

Fat Cells ◽

Adipose Derived Stem Cells ◽

Osteogenic Medium ◽

Alizarin Red ◽

Dedifferentiated Fat Cells ◽

Dfat Cells

Background: We investigated and compared the osteogenic potential and bone regeneration capacities of dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs). Method: We isolated DFAT cells and ASCs from GFP mice. DFAT cells were established by a new culture method using a mesh culture instead of a ceiling culture. The isolated DFAT cells and ASCs were incubated in osteogenic medium, then alizarin red staining, alkaline phosphatase (ALP) assays, and RT-PCR (for RUNX2, osteopontin, DLX5, osterix, and osteocalcin) were performed to evaluate the osteoblastic differentiation ability of both cell types in vitro. In vivo, the DFAT cells and ASCs were incubated in osteogenic medium for four weeks and seeded on collagen composite scaffolds, then implanted subcutaneously into the backs of mice. We then performed hematoxylin and eosin staining and immunostaining for GFP and osteocalcin. Results: The alizarin red-stained areas in DFAT cells showed weak calcification ability at two weeks, but high calcification ability at three weeks, similar to ASCs. The ALP levels of ASCs increased earlier than in DFAT cells and showed a significant difference (p < 0.05) at 6 and 9 days. The ALP levels of DFATs were higher than those of ASCs after 12 days. The expression levels of osteoblast marker genes (osterix and osteocalcin) of DFAT cells and ASCs were higher after osteogenic differentiation culture. Conclusion: DFAT cells are easily isolated from a small amount of adipose tissue and are readily expanded with high purity; thus, DFAT cells are applicable to many tissue-engineering strategies and cell-based therapies.

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Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth

Journal of Materials Science Materials in Medicine ◽

10.1007/s10856-020-06475-6 ◽

2021 ◽

Vol 32 (1) ◽

Author(s):

Mariane Beatriz Sordi ◽

Raissa Borges Curtarelli ◽

Izabella Thaís da Silva ◽

Gislaine Fongaro ◽

Cesar Augusto Magalhães Benfatti ◽

...

Keyword(s):

Stem Cells ◽

Extracellular Matrix ◽

Osteogenic Differentiation ◽

Culture Media ◽

Deciduous Teeth ◽

Free Calcium ◽

Alizarin Red ◽

Matrix Mineralization ◽

Media Supplementation

AbstractIn in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1—SHED + Dulbecco’s Modified Eagles’ Medium (DMEM) + fetal bovine serum (FBS); G2—SHED + DMEM + FBS + DEX; G3—SHED + DMEM + FBS + ASC + βGLY; G4—SHED + DMEM + FBS + ASC + βGLY + DEX; G5—MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

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200 TRANSPLANTATION AND IN VIVO DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN SWINE

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab200 ◽

2006 ◽

Vol 18 (2) ◽

pp. 208 ◽

Cited By ~ 1

Author(s):

A. S. Lima ◽

S. A. Malusky ◽

M. R. B. Mello ◽

S. J. Lane ◽

J. R. Rivera ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Cellular Response ◽

Agricultural Research ◽

Primary Concern ◽

Adipose Derived Stem Cells ◽

Subcutaneous Adipose

A primary concern in stem cell biology is that observations made in vitro may be an artifact of the in vitro culture environment. In vitro derived stem cells can be implanted into the environment from which they are derived so that their response to physiological conditions may be observed. Several important cellular characteristics need to be examined following the cell's reintroduction to the in vivo environment, including the potential for differentiation, proliferative ability, and life span. Studying implanted stem cells will assist in determining the potential for stem cell use in clinical therapies and provide further understanding of the role adult stem cells have in the adult body. Currently, the scientific literature is lacking a detailed description of the cellular response of adipose-derived stem cells (ADSCs) reintroduced to their exact tissue of origin. Thus, the aim of this study was to evaluate porcine ADSC growth in vivo and to analyze cell differentiation in vivo following injection of undifferentiated ADSCs into subcutaneous fat. Subcutaneous adipose tissue was isolated from the back fat of male pigs (11 months of age) and digested with 0.075% collagenase at 37�C for 90 min. The digested tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM and the ADSCs were plated onto 75 cm2 flasks (5000-10 000 cells per cm2) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 ADSCs were labeled with fluorescent dye (PKH26; Sigma, St. Louis, MO, USA) and sorted by flow cytometry. After sorting, positive cells were washed and re-suspended in culture medium. For transplantation, 100 �L of cell suspension in DMEM containing one of four cell concentrations (0 (control); 30 000; 300 000; and 900 000 cells) were placed in a 1-mL syringe and injected into the subcutaneous back fat of recipient pigs (n = 2). Each pig had previously been tattooed with 12 13 � 13 squares to mark injection sites. The treatments were replicated three times within each animal. Two and three weeks after transplantation, animals were euthanized, the back fat containing the transplantation site was harvested, and the cells were disaggregated as described above. The buoyant adipocytes and pelleted ADSCs cells were then analyzed by flow cytometry. The results indicated that there were dose- and time-dependent increases in labeled ADSCs and labeled adipocytes in the fat samples with increasing cell number (from 0 to 300 000 cells). There was, however, a decrease in labeled ADSCs at the 900 000-cell dose, which is likely due to excess cells being transplanted or an immune reaction. Both of these aspects are currently being evaluated. In conclusion, undifferentiated ADSCs from swine can be isolated from and returned to the subcutaneous adipose layer and differentiate into mature adipocytes. This work was supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois.

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189 The Effect of Vitamin K on the Differentiation of Adipose-Derived Stem Cells into Osteoblasts

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab189 ◽

2018 ◽

Vol 30 (1) ◽

pp. 234

Author(s):

T. A. Bane ◽

J. C. Bertels ◽

K. M. Polkoff ◽

M. Rubessa ◽

M. B. Wheeler

Keyword(s):

Stem Cells ◽

Vitamin K ◽

Bone Mineralization ◽

Negative Control ◽

Nodule Formation ◽

Adipose Derived Stem Cells ◽

Osteogenic Medium ◽

Mineral Density ◽

Alizarin Red ◽

Significant Difference

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. This involves using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules. The aim of this project was to evaluate the effects of vitamin K in osteogenic medium and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Vitamin K has been shown to increase bone mineral density by acting as a coenzyme in the γ-carboxylation of osteocalcin, a protein involved in bone mineralization (Weber 2001 Nutrition 11–12, 1024). Our hypothesis was that the presence of vitamin K in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided into 7 different treatments: 5 concentrations of vitamin K in the osteogenic medium (10, 50 100, 500, 1000 nM) plus 2 control treatments (osteogenic medium without vitamin K and a negative control, DMEM). The media was changed twice a week for 4 weeks. The experiment was replicated 6 times. At the end of the culture period, cells were stained with Alizarin Red S and Von Kossa. In each well, we counted the nodules and then divided them in 2 categories: formed and forming nodules. Data were analysed by analysis of variance using the generalized linear model (GLM) procedure of SPSS (IBM/SPSS, Armonk, NY, USA); the least significant difference (l.s.d.) post hoc test was used to perform statistical multiple comparison, and the α-level was set at 0.05. The results showed (in Table 1) that there was no positive effect on nodule formation when vitamin K was added to the medium; however, when 1000 nM vitamin K was added, nodule formation decreased. More experiments need to be conducted to determine if vitamin K can act synergistically with other vitamins to produce a significant role in ASC differentiation into osteoblasts. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with vitamin K incorporated into their matrix. Table 1.The average number of formed and forming osteoblast nodules compared between treatment groups (SD in parentheses)

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208 The effect of copper on the differentiation of adipose-derived stem cells into osteoblasts

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab208 ◽

2019 ◽

Vol 31 (1) ◽

pp. 229

Author(s):

T. Bane ◽

L. Siegel ◽

J. Bertels ◽

K. Ratz ◽

M. Rubessa ◽

...

Keyword(s):

Stem Cells ◽

Lysyl Oxidase ◽

Collagen Matrix ◽

Negative Control ◽

Nodule Formation ◽

Adipose Derived Stem Cells ◽

Osteogenic Medium ◽

Alizarin Red ◽

Significant Difference ◽

Positive Effect

Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules offer a potential solution to this difficult problem. The aim of this project was to evaluate whether osteogenic medium infused with copper influences the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Copper is a key cofactor for lysyl oxidase, an enzyme involved in producing a collagen matrix through which bone can grow. Lysyl oxidase expression is up-regulated in bone marrow stromal cells (Khosravi et al. 2014 PLoS One 9, e100669). Our hypothesis was that the presence of copper in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). The ASC were divided in 7 different treatments: 5 concentrations of copper in the osteogenic medium (0.1, 1, 10, 50, and 100 µM) plus 2 control treatments (osteogenic medium without copper and a negative control, DMEM). The medium was changed twice a week for 4 weeks. The experiment was replicated 6 times. At the end of the culture period, cells were stained with Alizarin Red S and Von Kossa stains. In each well, we counted the total number of nodules that were either formed or forming. Data were analysed using the generalized linear model (GLM) procedure (SPSS Inc./IBM Corp., Armonk, NY). The least significant difference (l.s.d.) post hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results showed that more nodules were formed in the 0.1 and 1 µM copper groups compared with the osteogenic control, but there was no statistical difference between those 2 treatments. Table 1 illustrates the total number of formed and forming nodules in addition to their standard deviation. There was a positive effect on nodule formation when copper concentrations of 0.1 and 1 µM were added to the osteogenic medium. In contrast, copper concentrations of 50 and 100 µM had a cytotoxic effect. These results confirm that low concentrations of copper have a positive effect on osteogenesis. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with copper incorporated into their matrix. Table 1.The average number (standard deviations in parentheses) of total formed and forming osteoblast nodules compared between treatment groups

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Depletion of SNRNP200 inhibits the osteo−/dentinogenic differentiation and cell proliferation potential of stem cells from the apical papilla

BMC Developmental Biology ◽

10.1186/s12861-020-00228-y ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Xiaomin Su ◽

Haoqing Yang ◽

Ruitang Shi ◽

Chen Zhang ◽

Huina Liu ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cell Proliferation ◽

S Phase ◽

Alizarin Red ◽

Differentiation Potential ◽

Alp Activity ◽

Proliferation And Differentiation ◽

Apical Papilla ◽

Dentinogenic Differentiation

Abstract Background Tissue regeneration mediated by mesenchymal stem cells (MSCs) is deemed a desirable way to repair teeth and craniomaxillofacial tissue defects. Nevertheless, the molecular mechanisms about cell proliferation and committed differentiation of MSCs remain obscure. Previous researches have proved that lysine demethylase 2A (KDM2A) performed significant function in the regulation of MSC proliferation and differentiation. SNRNP200, as a co-binding factor of KDM2A, its potential effect in regulating MSCs’ function is still unclear. Therefore, stem cells from the apical papilla (SCAPs) were used to investigate the function of SNRNP200 in this research. Methods The alkaline phosphatase (ALP) activity assay, Alizarin Red staining, and osteogenesis-related gene expressions were used to examine osteo−/dentinogenic differentiation potential. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) and cell cycle analysis were applied to detect the cell proliferation. Western blot analysis was used to evaluate the expressions of cell cycle-related proteins. Results Depletion of SNRNP200 caused an obvious decrease of ALP activity, mineralization formation and the expressions of osteo−/dentinogenic genes including RUNX2, DSPP, DMP1 and BSP. Meanwhile, CFSE and cell cycle assays revealed that knock-down of SNRNP200 inhibited the cell proliferation and blocked cell cycle at the G2/M and S phase in SCAPs. In addition, it was found that depletion of SNRNP200 up-regulated p21 and p53, and down-regulated the CDK1, CyclinB, CyclinE and CDK2. Conclusions Depletion of SNRNP200 repressed osteo−/dentinogenic differentiation potentials and restrained cell proliferation through blocking cell cycle progression at the G2/M and S phase, further revealing that SNRNP200 has crucial effects on preserving the proliferation and differentiation potentials of dental tissue-derived MSCs.

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A Novel Fragment Derived from Laminin-411 Facilitates Proliferation and Differentiation of Odontoblast-Like Cells

BioMed Research International ◽

10.1155/2018/9465383 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Jia Tang ◽

Takashi Saito

Keyword(s):

Proliferative Activity ◽

Mrna Level ◽

Cell Counting ◽

Rt Pcr ◽

Alizarin Red ◽

Alp Activity ◽

Proliferation And Differentiation ◽

Post Hoc ◽

Laminin 411

The aim for the present study was to evaluate the in vitro effects of iMatrix-411 in odontoblast-like cells. To that end, iMatrix-411 was coated to both nontissue culture treated- (Non-PS) and tissue culture treated-polystyrene (TCPS) multiwells. MDPC-23 cells were seeded into noncoated (control) or coated wells. Optimal coating density and cell proliferation were assessed by cell counting kit-8 (CCK-8) at day two, day three, and day five. Osteo/odontogenic differentiation was evaluated by real-time RT-PCR and alkaline phosphatase (ALP) activity at days seven and eight, respectively. Calcific deposition of cells was visualized by alizarin red staining. Data were analyzed with post hoc Tukey HSD test (p<0.05). Optimal coating density for iMatrix-411 was 8 μg/cm2. Exposure of MDPC-23 cells to iMatrix-411 in either non-PS or TCPS significantly enhanced proliferative activity. iMatrix-411 elevated ALP activity in both types of culture plates. iMatrix-411 significantly increased the mRNA level of OCN, BSP, OPN, ALP, and DMP-1. Meanwhile, it enhanced the expression of several integrin subunits: ITGA1, ITGA5, ITGAV, ITGB1, and ITGB5. Finally, iMatrix-411 also accelerated the mineralization at day eight in Non-PS. The results indicated iMatrix-411 stimulates proliferation and favours differentiation of odontoblast-like cells.

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Extracellular IL-37 Enhances Osteogenic and Odontogenic Differentiation of Human Dental Pulp Stem Cells via Autophagy Pathway

10.21203/rs.3.rs-291517/v1 ◽

2021 ◽

Author(s):

Na Li ◽

Yan Chen ◽

Ming Yan ◽

Yanqiu Wang ◽

Jintao Wu ◽

...

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Inflammatory Factor ◽

Alizarin Red ◽

Dental Tissues ◽

Alp Activity ◽

Odontogenic Differentiation ◽

Marrow Mesenchymal Stem Cells

Abstract BackgroundThe osteogenic and odontogenic differentiation of dental pulp stem cells (DPSCs) contributes to the restoration and regeneration of dental tissues. Previous study indicated that IL-37 has often been identified as an anti-inflammatory factor that affects other pro-inflammatory signals. It is known to be a factor capable of inducing in vitro osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). The aims of this study were to explore the effects of IL-37 on the differentiation of DPSCs.MethodsDPSCs were cultured in growth medium with different concentration of IL-37, ALP activity was done to detect the optimal concentration for the following experiments. CCK-8 were conducted to assess the effect of IL-37 on proliferation of DPSCs. To assess differentiation, alkaline phosphatase activity, ALP staining, alizarin red S staining and real‐time RT‐PCR of DSPP, Runx2, ALP, and OSX were measured. Western blot was conducted to examine the levels of autophagy related markers (Beclin1, P62, LC3). ResultsCells cultured with 1 ng/mL IL-37 owned the highest ALP activity. IL-37 enhanced the osteogenic and odontogenic differentiation of DPSCs following upregulated the expression of Beclin1, downregulated the expression of P62, and reduced the ratio of LC3II/I, whereas depletion of autophagy suppressed DPSCs osteogenic and odontogenic differentiation. ConclusionIL-37 increased osteogenic and odontogenic differentiation via autophagy.

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202 IN VITRO DIFFERENTIATION OF ADULT PORCINE ADIPOSE-DERIVED STEM CELLS AFTER LABELING WITH PKH26 AND FLOW CYTOMETRY

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab202 ◽

2006 ◽

Vol 18 (2) ◽

pp. 209

Author(s):

M. Mello ◽

A. Lima ◽

S. Malusky ◽

S. Lane ◽

M. Wheeler

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Adipose Tissue ◽

Osteogenic Differentiation ◽

Fluorescent Dye ◽

Positive Staining ◽

Adipose Derived Stem Cells ◽

Differentiation Potential ◽

Oil Red O

The purpose of this study was to investigate the possible effects of the fluorescent dye PKH26 and flow cytometry on adult porcine adipose-derived stem cells (ADSCs) after exposing them to adipogenic and osteogenic differentiation conditions. Adipose tissue was isolated from swine (11 months of age) and digested with 0.075% collagenase at 37�C for 90 min. The digested adipose tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM, and the ADSCs were plated onto 75 cm2 flasks (5000-10 000 cells per cm2) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 cells were labeled with fluorescent dye (PKH26 red fluorescent cell linker kit; Sigma Chemical, St. Louis, MO, USA) and sorted by flow cytometry. After labeling and sorting, the sorted and unsorted (control group) cells were replated and exposed to adipogenic (1 �M dexamethasone, 0.5 mM isobutylmethylxantine, 10 �M insulin and 200�M indomethacin) and osteogenic (0.1 �M dexamethasone, 10 mM �-glycerophosphate, and 50�M ascorbic acid) differentiation conditions when the cells were 90% confluent. Cells were evaluated based on morphology and specific staining properties. Adipogenic differentiation was confirmed by oil red O-positive staining of large lipid vacuoles, and osteogenic differentiation by Von Kossa staining 2 weeks after initiation of differentiation. The frequency of oil red O-positive colonies in both sorted and unsorted group was similar (15.0% vs. 13.2%, respectively). The number of osteogenic nodules, confirmed by the presence of calcium by Von Kossa staining, in the sorted and unsorted group was 17 and 184 per flask, respectively. In conclusion, this study demonstrates that adult porcine adipose-derived stem cells maintain their differentiation potential after labeling with fluorescent dye and sorting by flow cytometry. This should allow for more rapid evaluation of the differentiation potential of ADSCs in vitro. This work was partially supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois and CNPq, Brazil (M. Mello).

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