Higher maternal age is associated with higher occurrence of cleft lip/palate in neonates under intensive care

Aim: To assess the prevalence of cleft lip and/or cleft palate (CL/P) and associated variables in neonates admitted to neonatal intensive care units (ICU). Methods: Medical charts for neonates born and admitted to the ICU between 2012 and 2018 were reviewed. Obstetric and neonatal variables were collected by a trained researcher. In the case group, all neonates with CL/P were included. The control group was formed by matching sex, prematurity and month of birth using random number generation. Neonates with congenital malformations were excluded from the control group. Adjusted logistic regression was used (p<0.05). Results: The prevalence of CL/P was 0.43% (n=15). Five cases were excluded, as pairing was not possible. Twenty neonates were included in the control group. In the final multivariate model, CL/P was only associated with increased maternal age. For each year of increase in maternal age, neonates had a 35.2% higher chance of presenting CL/P (95% confidence interval: 1.021­1.792). Conclusions: Higher maternal age was associated with higher occurrence of CL/P in neonates admitted to the ICU. No other neonatal or maternal independent variables were associated with CL/P. Due to missing data, interpretation of study results must be approached with caution

Comparison of senescence phenotype of short- and long- term cultured rat mesenchymal stem cells in vitro.

Mesenchymal stem cells present clinical potential to recover and regenerate injured tissues in diverse pathologies. The in vitro expansion and characterization of these cells contribute to elucidation of the mechanisms of senescence and strategies involving cell therapies. This study aimed to compare specific characteristics between initial and advanced passages of mesenchymal stem cells derived from adipose tissue and bone marrow. Both cell types were characterized according to immunophenotype, osteogenic differentiation, genomic instability, migration assay, doubling population time and colony forming ability. Our results demonstrated that both cell types were able to maintain an immunophenotypic profile typical of mesenchymal stem cells during increasing passages. Adipose stem cells at initial passage presented greater migration capacity compared to advanced passage cells, and advanced passage cells proliferated faster than initial passage cells. Bone marrow stem cells at early passages presented higher osteogenic potential than advanced. At advanced passages they presented higher colony forming capacity and genetic damage than those at initial passage. These results suggest that mesenchymal stem cells maintained in culture presented characteristics of senescence that should be monitored prior the use in regenerative medicine and cells derived from bone marrow at initial passage have better potential for therapeutic use in bone tissue engineering.

Fluorosilicic acid induces DNA damage and oxidative stress in bone marrow mesenchymal stem cells.

Excess fluoride in water can produce changes in tooth enamel mineralization and lead to diseases such as dental or skeletal fluorosis. The present study aimed to assess the genotoxic effects, oxidative stress, and osteoblastic mineralization induced by fluorosilicic acid (FA) in murine bone marrow-derived mesenchymal stem cells (BM-MSCs). BM-MSCs were isolated from the femurs and tibias of rats and cultured under standard conditions. Cells exposure occurred for 3, 7, 14, and 21 days to different concentrations of FA (0.6-9.6 mg/L). Cytotoxicity was observed in 14 and 21 days of exposure for all concentrations of FA (cell proliferation below 60%), and for 3 and 7 days, in which the proliferation was above 80%. Alkaline comet assay results demonstrated significant increased damage at concentrations of 0.3-2.4 mg/L, and the micronucleus test showed increased rates for micronucleus (1.2-2.4 mg/L) and nuclear buds (NBUDs) (0.3-2.4 mg/L) (P < 0.05/Dunnett's test). An alkaline comet assay modified by repair endonuclease (FPG) was used to detect oxidized nucleobases, which occurred at 0.6 mg/L. The oxidative stress was evaluated by lipid peroxidation (TBARS) and antioxidant activity (TAC). Only lipid peroxidation was increased at concentrations of 0.6 mg/L and 1.2 mg/L (P < 0.001/Tukey's test). The osteogenesis process determined the level of extracellular matrix mineralization. The mean concentration of Alizarin red increased significantly in 14 days at the 0.6 mg/L concentration group (P < 0.05/Tukey's test) compared to the control group, and a significant difference between the groups regarding the activity of alkaline phosphatase (ALP) was observed. Unlike other studies, our results indicated that FA in BM-MSCs at concentrations used in drinking water induced genotoxicity, oxidative stress, and acceleration of bone mineralization.

Combining canine mesenchymal stromal cells and hyaluronic acid for cartilage repair.

Cell therapy and tissue engineering have been intensively researched for repair of articular cartilage. In this study, we investigated the chondrogenic potential of canine adipose-derived mesenchymal stromal cells (ASCs) combined to high molecular weight hyaluronic acid (HA) in vitro, and their therapeutic effect in dogs with chronic osteoarthritis (OA) associated with bilateral hip dysplasia. Canine ASCs were characterized after conventional 2D culture or 3D culture in HA, showing adequate immunophenotype, proliferation and trilineage differentiation, as well as chondrogenesis after cultivation in HA. ASC/HA constructs were used to treat 12 dogs with OA, sequentially assigned to control, ASC and ASC/HA groups. Animals were examined for clinical, orthopedic and radiological parameters. Lameness at walk and pain on manipulation were reduced in the ASC group and mainly in the ASC/HA group. Range of motion and detection of crepitus on hip rotation and abduction improved similarly in all groups. For articular edema, muscle atrophy, Norberg angle values and radiographic analyses, there were no variations throughout the period. These results indicate that ASC/HA constructs are safe and may be an effective therapeutic tool in treating canine chronic osteoarthritis, which should be confirmed with larger studies and additional clinical parameters.

Improvement of human pancreatic islet quality after co-culture with human adipose-derived stem cells.

The aim of this study was to investigate whether co-culture of human islets with adipose-derived stem cells (ASCs) can improve islet quality and to evaluate which factors play a role in the protective effect of ASCs against islet dysfunction. Islets and ASCs were cultured in three experimental groups for 24 h, 48 h, and 72 h: 1) indirect co-culture of islets with ASC monolayer (Islets/ASCs); 2) islets alone; and 3) ASCs alone. Co-culture with ASCs improved islet viability and function in all culture time-points analyzed. VEGFA, HGF, IL6, IL8, IL10, CCL2, IL1B, and TNF protein levels were increased in supernatants of islet/ASC group compared to islets alone, mainly after 24 h. Moreover, VEGFA, IL6, CCL2, HIF1A, XIAP, CHOP, and NFKBIA genes were differentially expressed in islets from the co-culture condition compared to islets alone. In conclusion, co-culture of islets with ASCs promotes improvements in islet quality.

Osteogenic differentiation of bone marrow-derived mesenchymal stem cells on anodized niobium surface.

Currently, titanium and its alloys are the most used materials for biomedical applications. However, because of the high costs of these metals, new materials, such as niobium, have been researched. Niobium appears as a promising material due to its biocompatibility, and excellent corrosion resistance. In this work, anodized niobium samples were produced and characterized. Their capacity to support the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) was also tested. The anodized niobium samples were characterized by SEM, profilometry, XPS, and wettability. BM-MSCs were cultured on the samples during 14 days, and tested for cell adhesion, metabolic activity, alkaline phosphatase activity, and mineralization. Results demonstrated that anodization promotes the formation of a hydrophilic nanoporous oxide layer on the Nb surface, which can contribute to the increase in the metabolic activity, and in osteogenic differentiation of BM-MSCs, as well as to the extracellular matrix mineralization.

Calcitriol combined with calcium chloride causes apoptosis in undifferentiated adipose tissue-derived human mesenchymal stem cells, but this effect decreases during adipogenic differentiation.

Calcitriol, the bioactive hormone of vitamin D, is currently linked to several diseases, such as obesity and gain of adipose mass, due to its liposolubility and, consequently, its sequestration by adipocytes. As rates of obesity continue to increase, research on the biology of weight gain should be encouraged. This study evaluated the effects of calcitriol combined with CaCl2 on adipose tissue-derived human mesenchymal stem cells. We evaluated the cytotoxicity of the combination by MTT assays, in which undifferentiated cells and cells undergoing adipogenic differentiation were tested for 7 and 14 days. The results demonstrated that the combination of calcitriol at the IC50 and CaCl2 at the IC20 was effective at reducing the viability of mesenchymal stem cells, but with the progression of cell differentiation towards adipocytes, cell resistance to the cytotoxic effects increased. The percentages of dead cells were 88.29, 57.45 and 28.81% for undifferentiated cells and cells exposed to differentiation medium for 7 and 14 days, respectively. Undifferentiated cells were evaluated for apoptosis in response to the same combination using Annexin V assays, and a possible onset of programmed cell death in undifferentiated cells was detected. Additionally, the combination of the compounds altered the membrane permeability of undifferentiated cells by 16 percentage points and induced cell cycle arrest in S phase due to the accumulation of damage. An evaluation of gene expression revealed the overexpression of the GADD45 and ATM genes and the underexpression of the P21, P53, ATR, BCL-2, EIF2 AK3, IGF1R, DNAse-2, ATF, MAP3K4, ENGO-G, CASP3, CASP7 and CASP8 genes. Our results provide valuable insights into the biology of obesity and may contribute to the development of new anti-obesity therapies focusing on the inhibition of adipose tissue mesenchymal stem cell hyperplasia and adipogenic differentiation.

Vitamin D: Correlation with biochemical and body composition changes in a southern Brazilian population and induction of cytotoxicity in mesenchymal stem cells derived from human adipose tissue.

Studies have shown that metabolic disorders, serum inflammatory markers and weight gain (obesity) are correlated with vitamin D deficiency. Therefore, the present study correlated the serum calcidiol (s25(OH)D3) levels in a sample of individuals from southern Brazil with variables related to metabolic disorders, obesity and lifestyle habits and assessed the cytotoxic effect of calcitriol on adipose tissue-derived mesenchymal stem cells (ADSCs). The results showed a 79.23% prevalence of hypovitaminosis D in the study population and a correlation (p<0.05) between a low serum vitamin D concentration and an elevated low-density lipoprotein cholesterol (LDL-c) level. Univariate linear regression analysis using 25(OH)D3 as a regressor showed a negative association (p<0.05) with an indoor work environment (ß=-2.305), increased body fat (ß=-0.095), age (ß=-0.065) and high-density lipoprotein cholesterol (HDL-c; ß=-0.109). An in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay performed with ADSCs using five calcitriol concentrations (15.625, 31.25, 62.5, 125 and 250nM) indicated cytotoxic potential (p<0.05) at the 62.5nM concentration at 48 and 72h and at the 125 and 250nM concentrations at 24, 48 and 72h. The results reported herein corroborate one another and suggest a key association between vitamin D deficiency and the development of obesity because ADSCs are involved in adipose tissue hyperplasia and differentiate into adipocytes that can sequester the bioavailable vitamin D necessary for homeostasis.

Repair of bone defects using adipose-derived stem cells combined with alpha-tricalcium phosphate and gelatin sponge scaffolds in a rat model.

OBJECTIVES: This study aimed to evaluate the potential of adipose-derived stem cells (ASCs) combined with a modified α-tricalcium phosphate (α-TCP) or gelatin sponge (GS) scaffolds for bone healing in a rat model. MATERIAL AND METHODS: Bone defects were surgically created in the femur of adult SHR rats and filled with the scaffolds, empty or combined with ASCs. The results were analyzed by histology and histomorphometry on days seven, 14, 30, and 60. RESULTS: Significantly increased bone repair was observed on days seven and 60 in animals treated with α-TCP/ASCs, and on day 14 in the group treated with GS/ASCs, when compared with the groups treated with the biomaterials alone. Intense fibroplasia was observed in the group treated with GS alone, on days 14 and 30. CONCLUSIONS: Our results showed that the use of ASCs combined with α-TCP or GS scaffolds resulted in increased bone repair. The higher efficacy of the α-TCP scaffold suggests osteoconductive property that results in a biological support to the cells, whereas the GS scaffold functions just as a carrier. These results confirm the potential of ASCs in accelerating bone repair in in vivo experimental rat models. These results suggest a new alternative for treating bone defects.

Repair of bone defects using adipose-derived stem cells combined with alpha-tricalcium phosphate and gelatin sponge scaffolds in a rat model

Abstract Objectives This study aimed to evaluate the potential of adipose-derived stem cells (ASCs) combined with a modified α-tricalcium phosphate (α-TCP) or gelatin sponge (GS) scaffolds for bone healing in a rat model. Material and Methods Bone defects were surgically created in the femur of adult SHR rats and filled with the scaffolds, empty or combined with ASCs. The results were analyzed by histology and histomorphometry on days seven, 14, 30, and 60. Results Significantly increased bone repair was observed on days seven and 60 in animals treated with α-TCP/ASCs, and on day 14 in the group treated with GS/ASCs, when compared with the groups treated with the biomaterials alone. Intense fibroplasia was observed in the group treated with GS alone, on days 14 and 30. Conclusions Our results showed that the use of ASCs combined with α-TCP or GS scaffolds resulted in increased bone repair. The higher efficacy of the α-TCP scaffold suggests osteoconductive property that results in a biological support to the cells, whereas the GS scaffold functions just as a carrier. These results confirm the potential of ASCs in accelerating bone repair in in vivo experimental rat models. These results suggest a new alternative for treating bone defects.

Identification of suitable reference genes for quantitative gene expression analysis in rat adipose stromal cells induced to trilineage differentiation.

This study was designed to (i) identify stable reference genes for the analysis of gene expression during in vitro differentiation of rat adipose stromal cells (rASCs), (ii) recommend stable genes for individual treatment conditions, and (iii) validate these genes by comparison with normalization results from stable and unstable reference genes. On the basis of a literature review, eight genes were selected: Actb, B2m, Hprt1, Ppia, Rplp0, Rpl13a, Rpl5, and Ywhaz. Genes were ranked according to their stability under different culture conditions as assessed using GenNorm, NormFinder, and RefFinder algorithms. Although the employed algorithms returned different rankings, the most frequently top-ranked genes were: B2m and/or Ppia for all 28day treatments (ALL28); Ppia and Hprt1 (adipogenic differentiation; A28), B2m (chondrogenic differentiation; C28), Rpl5 (controls maintained in complete culture medium; CCM), Rplp0 (osteogenic differentiation for 3days; O3), Rpl13a and Actb (osteogenic differentiation for 7days; O7), Rplp0 and Ppia (osteogenic differentiation for 14days; O14), Hprt1 and Ppia (osteogenic differentiation for 28days; O28), as well as Actb (all osteogenesis time points combined; ALLOSTEO). The obtained results indicate that the performance of reference genes depends on the differentiation protocol and on the analysis time, thus providing valuable information for the design of RT-PCR experiments.

Human mesenchymal stem cells are resistant to cytotoxic and genotoxic effects of cisplatin in vitro.

Mesenchymal stem cells (MSCs) are known for their important properties involving multilineage differentiation potential., trophic factor secretion and localization along various organs and tissues. On the dark side, MSCs play a distinguished role in tumor microenvironments by differentiating into tumor-associated fibroblasts or supporting tumor growth via distinct mechanisms. Cisplatin (CIS) is a drug widely applied in the treatment of a large number of cancers and is known for its cytotoxic and genotoxic effects, both in vitro and in vivo. Here we assessed the effects of CIS on MSCs and the ovarian cancer cell line OVCAR-3, by MTT and comet assays. Our results demonstrated the resistance of MSCs to cell death and DNA damage induction by CIS, which was not observed when OVCAR-3 cells were exposed to this drug.

Mesenchymal stem cells and their relationship to pericytes.

Our body contains cells that can be propagated in vitro and give rise to cells with mature mesenchymal phenotypes. These cells are interesting not only because of their differentiation capability, which could be used for tissue engineering, but also because they secrete molecules which have trophic, chemoattractant, and immunomodulatory properties. Along decades of study, these cells have been referred to as fibroblastic cells, stromal cells, or mesenchymal stem cells. There is evidence that pericytes, cells that wrap endothelial cells in blood vessels, behave as stem cells in the tissues, and give rise to these progenitor cells when removed from the body and expanded in culture - a process that may reflect changes that occur in vivo under injury conditions. Here, we discuss the evidence that favors this thesis, and discuss culture methods, clinical and preclinical applications of mesenchymal stem cells under this perspective.

Gelatin and galactomannan-based scaffolds: Characterization and potential for tissue engineering applications.

In this work, we produced gelatin films containing different concentrations of galactomannan by casting solutions. The films were crosslinked by immersion in 30mM solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). The crosslinking of gelatin-containing films was confirmed by the reduction of free amine band intensity (3400-3200cm(-1)) in the GEL IR, as well as by the evaluation of its behavior when immersed in phosphate-buffer solution. The crosslinking of galactomannan film was confirmed by the formation of new ether bonds, as observed by increasing intensity of the band at 1148cm(-1), and the reduction of OH band intensity (3600-3200cm(-1)). The presence of galactomannan and the crosslinking mediated by EDC were responsible to improve elasticity in the gelatin-based films. The samples did not show cytotoxicity during 24h or 48h. In addition, rat mesenchymal stem cells adhered to the films regardless of galactomannan concentration. The results indicated that the gelatin/galactomannan films are potential biomaterials for use as scaffolds for tissue engineering.

Desfechos materno-fetais do diabetes gestacional em serviço terciário de atenção obstétrica / Maternal and fetal outcomes of gestational diabetes in a tertiary obstetric center

Introdução: Diabetes mellitus gestacional (DMG) é uma das principais complicações da gestação, sendo definida como intolerância à glicose reconhecida neste período. Considerando as implicações neonatais e maternas decorrentes dessa comorbidade, este estudo tem como objetivo descrever os desfechos materno-fetais de gestantes diabéticas. Métodos: Estudo descritivo, em que se investigou o prontuário de 86 gestantes internadas por DMG (e seus neonatos), entre 01/01/2012 e 30/08/2013, no Hospital Universitário da Universidade Luterana do Brasil (ULBRA). Resultados: A média de idade foi 31,9 ± 6,9 anos. Quanto ao parto, 60,5% foi por cesárea, sendo a idade gestacional média de 37 semanas e 6 dias. 55,8% das gestantes apresentaram algum desfecho adverso. O peso médio dos recém nascidos foi 3416 ± 528,2 gramas, 20,9% apresentaram hipoglicemia, 3,5% hiperbilirrubinemia, 12,8% disfunção respiratória e 10,5% necessitaram UTI Neonatal. As incidências de prematuridade e recém-nascido grande para idade gestacional foram de 25,5%. O tempo médio de internação foi de 67 horas. Conclusões: Os resultados suportam os achados esperados de morbidade neonatal associada ao DMG, com expressiva incidência de desfechos neonatais constatados na população estudada, em especial, prematuridade, hipoglicemia neonatal e recém-nascido grande para a idade gestacional. Nossos dados reforçam a importância do rastreamento e manejo adequado desta patologia devido à alta incidência de complicações neonatais e morbidade associado a essa condição (AU)

Introduction: Gestational diabetes mellitus (GDM) is a major complication of pregnancy, defined as glucose intolerance recognized in this period. Considering the neonatal and maternal implications of this comorbidity, this study aims to describe the maternal and fetal outcomes of pregnant diabetic women. Methods: Descriptive study, which investigated the medical records of 86 pregnant women hospitalized for DMG (and their newborns) between Jan 1, 2012 and Aug 30, 2013 at the University Hospital of the Lutheran University of Brazil (ULBRA). Results: Mean age was 31.9 ± 6.9 years. As for the birth, 60.5% was by caesarean section, with mean gestational age of 37 weeks and 6 days. 55.8% of the patients presented some adverse outcome. The mean weight of newborns was 3416 ± 528.2 grams, 20.9% had hypoglycemia, 3.5%, hyperbilirubinemia, 12.8% respiratory dysfunction, and 10.5% required neonatal ICU. The incidence of prematurity and large-for-gestational-age newborn was 25.5%. The mean hospital stay was 67 hours. Conclusion: Our results support the expected findings of neonatal morbidity associated with GDM, with a significant incidence of neonatal outcomes observed in the study population, especially prematurity, neonatal hypoglycemia and large-for-gestational-age newborn. Our data reinforce the importance of screening and appropriate management of this disease due to the high incidence of neonatal morbidity and complications associated with this condition

Stability of Reference Genes during Tri-Lineage Differentiation of Human Adipose-Derived Stromal Cells.

Quantitative real-time PCR can detect variations in gene expression. The identification of the stable reference genes (RGs) is necessary to evaluate the expression of specific genes of interest under various conditions in many cell types, including human adipose-derived stromal cells (hASCs). In this study, we used the algorithms BestKeeper, NormFinder, geNorm, and RefFinder to investigate the stability of 15 potential RGs (B2M, eEF1A1, GAPDH, H2AFZ, HMBS, HPRT1, PGK1, PPIA, RPL5, SDHA, TBP, TKT, TRFC, TUBB, and UBC) in hASCs during control, adipo-, chondro-, and osteogenic differentiation for 28 days. RPL5, GAPDH, H2AFZ, and HPRT1 were the most stable RGs, while B2M and UBC were the least stable RGs for the majority of group analyses (tri-lineage differentiation and control analyzed combined or individually; each lineage combined with the control). These RGs were used to normalize adipo- (FABP4, LPL, and PPARG), chondro- (COMP and SOX9), and osteogenic gene expression markers (BMP4, COL1A1, and RUNX2). Each marker showed a similar expression when normalized by H2AFZ, HPRT1, or RPL5, confirming that these RGs exhibit stable expression. However, GAPDH, B2M, and UBC exhibited high standard deviation (SD), down-regulated and/or up-regulated differentiation gene expression markers when compared with stable RGs, demonstrating that these RGs are unstable.

Acupoint injection of autologous stromal vascular fraction and allogeneic adipose-derived stem cells to treat hip dysplasia in dogs.

Stem cells isolated from adipose tissue show great therapeutic potential in veterinary medicine, but some points such as the use of fresh or cultured cells and route of administration need better knowledge. This study aimed to evaluate the effect of autologous stromal vascular fraction (SVF, n = 4) or allogeneic cultured adipose-derived stem cells (ASCs, n = 5) injected into acupuncture points in dogs with hip dysplasia and weak response to drug therapy. Canine ASCs have proliferation and differentiation potential similar to ASCs from other species. After the first week of treatment, clinical evaluation showed marked improvement compared with baseline results in all patients treated with autologous SVF and three of the dogs treated with allogeneic ASCs. On days 15 and 30, all dogs showed improvement in range of motion, lameness at trot, and pain on manipulation of the joints, except for one ASC-treated patient. Positive results were more clearly seen in the SVF-treated group. These results show that autologous SVF or allogeneic ASCs can be safely used in acupoint injection for treating hip dysplasia in dogs and represent an important therapeutic alternative for this type of pathology. Further studies are necessary to assess a possible advantage of SVF cells in treating joint diseases.

Mesenchymal stem cells combined with an artificial dermal substitute improve repair in full-thickness skin wounds.

Autografts represent the gold standard for the treatment of full thickness burns. Factors such as lack of suitable donor sites and poor skin quality, however, have led to the development of artificial dermal substitutes. The investigation of mechanisms leading to enhanced functionality of these skin substitutes has been attracting great attention. This study aimed to investigate the effect of autologous stem cells on the integration and vascularization of a dermal substitute in full-thickness skin wounds, in a murine model. Two cell populations were compared, whole bone marrow cells and cultivated mesenchymal stem cells, isolated from mice transgenic for the enhanced green fluorescent protein, which allowed tracking of the transplanted cells. The number of cells colonizing the dermal substitute, as well as vascular density, were higher in mice receiving total bone marrow and particularly mesenchymal stem cells, than in control animals. The effect was more pronounced in animals treated with mesenchymal stem cells, which located primarily in the wound bed, suggesting a paracrine therapeutic mechanism. These results indicate that combining mesenchymal stem cells with artificial dermal substitutes may represent an important potential modality for treating full thickness burns, even in allogeneic combinations due to the immunoregulatory property of these cells.

Methodology, biology and clinical applications of human mesenchymal stem cells.

Stem cells are known by their capacity of self-renewal and differentiation into at least one specialized cell type. Mesenchymal stem cells (MSCs) were isolated initially from bone marrow but are now known to exist in any vascularized organ or tissue in adults. MSCs have a great therapeutic potential, due to their ability to migrate to sites of tissue injury and secrete trophic factors that hasten endogenous repair. They have also been shown to present immunosuppressive properties that may be used in the treatment of autoimmune or graft-versus-host diseases. Clinical trials employing MSCs show that the therapy is safe, but the efficiency needs to be in tested in phase III and IV studies. We describe here protocols for the isolation of human MSCs from human bone marrow and adipose tissue. The safe use of these cells demand a thorough in vitro characterization, as described in protocols of immunophenotyping by flow cytometry and analysis of their capacity to differentiate into adipogenic, osteogenic, and chondrogenic lineages.