Culture-Expanded Autologous Adipose-Derived Mesenchymal Stem Cell Treatment for Osteonecrosis of the Femoral Head.

BACKGROUD: Outcomes of traditional treatment for osteonecrosis of the femoral head (ONFH) are not always satisfactory. Hence, cell-supplementation therapy has been attempted to facilitate necrotic-tissue regeneration. Adipose-derived mesenchymal stem cell (ADMSC) transplantation is potentially advantageous over bone marrow-derived MSC implantation, but its outcomes for ONFH remain unclear. The aim of this study was to determine 2-year radiological and clinical outcomes of culture-expanded autologous ADMSC implantation for ONFH. METHODS: Eighteen hips with necrotic lesions involving ≥ 30% of the femoral head were included. ADMSCs were harvested by liposuction and culture expanded for 3 passages over 3 weeks. With a 6-mm single drilling, ADMSCs were implanted into the necrotic zone. All patients underwent magnetic resonance imaging (MRI), single-photon emission computed tomography/computed tomography (SPECT/CT) at screening and 6 months, 12 months, and 24 months postoperatively. The primary outcome was the change in the size of necrotic area on MRI. Secondary outcomes were changes in clinical scores and radioisotope uptake on SPECT/CT. Conversion total hip arthroplasty (THA) was defined as the endpoint. RESULTS: Preoperatively, the necrotic lesion extent was 63.0% (38.4%-96.7%) of the femoral head. The mean Harris hip score was 89.2, the University of California at Los Angeles (UCLA) score was 5.6, and Western Ontario and McMaster Universities Arthritis index (WOMAC) was 79.4. Three patients underwent THA and 1 patient died in an accident. Finally, 11 patients (14 hips) were available for ≥ 2-year follow-up. At the last follow-up, no surgery-related complications occurred, and 14 of 17 hips (82%) were able to perform daily activities without THA requirement. There was no significant decrease in lesion size between any 2 intervals on MRI. However, widening of high signal intensity bands on T2-weighted images inside the necrotic lesion was observed in 9 of 14 hips (64%); 11 of 14 hips (79%) showed increased vascularity on SPECT/CT at 2 years postoperatively. No significant differences were observed between preoperative and 24-month mean Harris hip score (89.2 vs. 88.6), WOMAC (79.4 vs. 75.7), and UCLA score (5.6 vs. 6.2). CONCLUSIONS: Our outcomes suggest that culture-expanded ADMSC implantation is a viable option for ONFH treatment without adverse events.

Comparison of Anti-Oxidative Effect of Human Adipose- and Amniotic Membrane-Derived Mesenchymal Stem Cell Conditioned Medium on Mouse Preimplantation Embryo Development.

Oxidative stress is a major cause of damage to the quantity and quality of embryos produced in vitro. Antioxidants are usually supplemented to protect embryos from the suboptimal in vitro culture (IVC) environment. Amniotic membrane-derived mesenchymal stem cells (AMSC) have emerged as a promising regenerative therapy, and their paracrine factors with anti-oxidative effects are present in AMSC conditioned medium (CM). We examined the anti-oxidative potential of human AMSC-CM treatment during IVC on mouse preimplantation embryo development and antioxidant gene expression in the forkhead box O (FoxO) pathway. AMSC-CM (10%) was optimal for overall preimplantation embryo developmental processes and upregulated the expression of FoxOs and their downstream antioxidants in blastocysts (BL). Subsequently, compared to adipose-derived mesenchymal stem cell (ASC)-CM, AMSC-CM enhanced antioxidant gene expression and intracellular GSH levels in the BL. Total antioxidant capacity and SOD activity were greater in AMSC-CM than in ASC-CM. Furthermore, SOD and catalase were more active in culture medium supplemented with AMSC-CM than in ASC-CM. Lastly, the anti-apoptotic effect of AMSC-CM was observed with the regulation of apoptosis-related genes and mitochondrial membrane potential in BL. In conclusion, the present study established AMSC-CM treatment at an optimal concentration as a novel antioxidant intervention for assisted reproduction.

Anti-Oxidative Effects of Human Adipose Stem Cell Conditioned Medium with Different Basal Medium during Mouse Embryo In Vitro Culture.

The quality of embryos produced by assisted reproductive techniques should be advanced by the improvement of in vitro culture conditions for successful implantation and pregnancy maintenance. We investigated the anti-oxidative effect of human adipose stem cell (ASC) conditioned medium with its optimal basal medium, Dulbecco's modified Eagle's medium (DMEM-CM), or keratinocyte serum-free medium (KSFM-CM) as supplements during in vitro culture (IVC) of in vitro fertilized mouse embryo. At first, preimplantation embryo development was evaluated in KSFM-CM and DMEM-CM supplemented cultures at various concentrations. The blastocyst (BL) and hatched BL formation rates were significantly increased in 5% DMEM-CM, while no difference was observed from KSFM-CM. Next, comparing the efficacy of KSFM-CM and DMEM-CM at the same concentration, DMEM-CM enhanced the developmental rate of 16 cells, morula, BL, and hatched BL. The expression level of reactive oxygen species decreased and that of glutathione increased in BL cultured with DMEM-CM, which confirms its anti-oxidative effect. Furthermore, apoptosis in BL cultured with DMEM-CM was reduced compared with that in KSFM-CM. This study demonstrated that the comparative effect of human ASC-CM made of two different basal media during mouse embryo IVC and anti-oxidative effect of 5% DMEM-CM was optimal to improve preimplantation embryo development.

High Frequency of Intravenous Injection of Human Adipose Stem Cell Conditioned Medium Improved Embryo Development of Mice in Advanced Maternal Age through Antioxidant Effects.

Advanced maternal age (AMA) has become prevalent globally. With aging, weakened antioxidant defense causes loss of normal function in the ovary and uterus due to oxidative stress. Here, we aimed to improve embryo development in AMA mice by intravenous injection (IV) of human adipose stem cell conditioned medium (ASC-CM) at various frequencies and intervals as an antioxidant intervention. Four- and six-month-old female ICR (Institute of Cancer Research) mice were randomly divided into groups IV treated with human ASC-CM under different conditions, and in vitro and in vivo embryo development were evaluated. Consequently, compared to the control group, blastocyst formation rate of parthenotes was significantly promoted in 4-month-old mice and the mean number of implanted fetuses after natural mating was significantly increased by approximately two-fold in 6-month-old mice. Through gene analysis, the anti-apoptotic and anti-oxidative effects of human ASC-CMs were confirmed in the ovaries and uterus of pregnant mice at both ages. In particular, ovarian expression of gpx1 and catalase drastically increased in 6-month-old mice. Furthermore, the levels of gpx1 and catalase were further increased, with a high frequency of injection regardless of age. Thus, we demonstrated for the first time the anti-oxidative effect of human ASC-CM administration against ovarian aging and the optimal injection condition.

Anti-osteoarthritis effect of a combination treatment with human adipose tissue-derived mesenchymal stem cells and thrombospondin 2 in rabbits.

BACKGROUND: Osteoarthritis (OA), a chronic age-related disease characterized by the slowly progressive destruction of articular cartilage, is one of the leading causes of disability. As a new strategy for treatment of OA, mesenchymal stem cells (MSCs) have the potential for articular cartilage regeneration. Meanwhile, thrombospondin 2 (TSP2) promotes the chondrogenic differentiation of MSCs. AIM: To investigate whether TSP2 induces chondrogenic differentiation of human adipose-derived MSCs (hADMSCs) and potentiates the therapeutic effects of hADMSCs in OA rabbits. METHODS: We investigated the chondrogenic potential of TSP2 in hADMSCs by analyzing the expression of chondrogenic markers as well as NOTCH signaling genes in normal and TSP2 small interfering RNA (siRNA)-treated stem cells. Anterior cruciate ligament transection surgery was performed in male New Zealand white rabbits, and 8 wk later, hADMSCs (1.7 × 106 or 1.7 × 107 cells) were injected into the injured knees alone or in combination with intra-articular injection of TSP2 (100 ng/knee) at 2-d intervals. OA progression was monitored by gross, radiological, and histological examinations. RESULTS: In hADMSC culture, treatment with TSP2 increased the expression of chondrogenic markers (SOX9 and collagen II) as well as NOTCH signaling genes (JAGGED1 and NOTCH3), which were inhibited by TSP2 siRNA treatment. In vivo, OA rabbits treated with hADMSCs or TSP2 alone exhibited lower degree of cartilage degeneration, osteophyte formation, and extracellular matrix loss 8 wk after cell transplantation. Notably, such cartilage damage was further alleviated by the combination of hADMSCs and TSP2. In addition, synovial inflammatory cytokines, especially tumor-necrosis factor-α, markedly decreased following the combination treatment. CONCLUSION: The results indicate that TSP2 enhances chondrogenic differentiation of hADMSCs via JAGGED1/NOTCH3 signaling, and that combination therapy with hADMSCs and TSP2 exerts synergistic effects in the cartilage regeneration of OA joints.

Clinical Assessment of Intravenous Endothelial Progenitor Cell Transplantation in Dogs.

Endothelial progenitor cells (EPCs) have been applied for cell therapy because of their roles in angiogenesis and neovascularization in ischemic tissue. However, adverse responses caused by EPC therapy have not been fully investigated. In this study, a human peripheral blood sample was collected from a healthy donor and peripheral blood mononuclear cells were separated using Ficoll-Hypaque. There were four experimental groups: 10 ml saline infusion group (injection rate; 3 ml/min), 10 ml saline bolus group (injection rate; 60 ml/min), 10 ml EPCs infusion group (2 x 105 cells/ml, injection rate; 3 ml/min), 10 ml EPCs bolus group (2 × 105 cells/ml, injection rate; 60 ml/min). Clinical assessment included physical examination and laboratory examination for intravenous human EPC transplantation in dogs. The results revealed no remarkable findings in vital signs among the dogs used. In blood analysis, platelet counts in saline infusion groups were significantly higher than in the EPC groups within normal ranges, and no significant differences were observed except K+, Cl- and blood urea nitrogen/urea. In ELISA assay, no significant difference was observed in serum tumor necrosis factor alpha. The serum concentration of vascular endothelial growth factor was significantly higher in EPC groups than in saline groups, and interleukin 10 was significantly up-regulated in the EPC infusion group compared with other groups. In conclusion, we demonstrated that no clinical abnormalities were detected after intravenous transplantation of human EPCs in dogs. The transplanted xenogenic EPCs might be involved in anti-inflammatory and angiogenic functions in dogs.

Intravenous human endothelial progenitor cell administration into aged mice enhances embryo development and oocyte quality by reducing inflammation, endoplasmic reticulum stress and apoptosis.

Stem cell therapy has been proposed to restore the function and structure of injured tissues. In the present study, we investigated the ability of human endothelial progenitor cells (hEPCs) to attenuate ovarian aging and dysfunction. Female ICR mice aged 4 and 6 months were injected with cultured hEPCs. Cultured hEPCs were injected intravenously twice with 5 × 104 cells with a 4 day interval. After pregnant mare serum gonadotropin and human chorionic gonadotropin stimulation, oocytes and ovaries of aged mice were collected, cumulus-free oocytes were activated by SrCl2 and gene expression levels related to inflammation, apoptosis, follicle development and endoplasmic reticulum (ER) stress in ovaries were compared. Administration of hEPCs attenuated the level of inflammatory cytokines and adverse apoptotic factor, as well as reducing ER stress in the ovaries. Increased cleavage and blastocyst formation rates and cell numbers in blastocysts from hEPCs-treated aged mice vs. same aged control mice demonstrated a protective function of hEPCs against reproductive aging. Based on these data, we suggest that treatment with hEPCs attenuates reproductive aging and dysfunction potentially via regulation of inflammation, apoptosis and ER stress.

Clinical assessment after human adipose stem cell transplantation into dogs.

Adipose tissue-derived stem cell (ASCs) are an attractive source of stem cells with therapeutic applicability in various fields for regenerating damaged tissues because of their stemness characteristics. However, little has reported on evaluating adverse responses caused by human ASC therapy. Therefore, in the present study, a clinical assessment after human ASC transplantation into dogs was undertaken. A total of 12 healthy male dogs were selected and divided into four groups: saline infusion, saline bolus, ASC infusion, and ASC bolus groups. Physical assessment and blood analysis were performed following ASC transplantation, and the concentrations of angiogenic factors, and pro- and anti-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). There were no adverse vital sign responses among the dogs. Blood analyses revealed no remarkable complete blood count or serum chemistry results. ELISA results for angiogenic and anti-inflammatory factors including matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were significantly higher in the two ASCs groups than in the controls. In conclusion, this study demonstrated that transplantation of human ASCs produced no adverse effects and could be used safely in dogs. In addition, human ASCs could be involved in modulating secretions of angiogenic factors including MMP9, VEGF, bFGF, and HGF and anti-inflammatory factor IL-10.

Correction: Enhanced Hepatogenic Transdifferentiation of Human Adipose Tissue Mesenchymal Stem Cells by Gene Engineering with Oct4 and Sox2.

[This corrects the article DOI: 10.1371/journal.pone.0108874.].

A Prospective, Nonrandomized, no Placebo-Controlled, Phase I/II Clinical Trial Assessing the Safety and Efficacy of Intramuscular Injection of Autologous Adipose Tissue-Derived Mesenchymal Stem Cells in Patients With Severe Buerger's Disease.

Buerger's disease is a rare and severe disease affecting the blood vessels of the limbs. Adipose tissue-derived mesenchymal stem cells (ADSCs) have the potential to cure Buerger's disease when developed as a stem cell drug. In the present study, we conducted a prospective, nonrandomized, no placebo-controlled, phase I/II clinical trial with a 2-year follow-up questionnaire survey. A total of 17 patients were intramuscularly administered autologous ADSCs at a dose of 5 million cells/kg. The incidence of adverse events (AEs), adverse drug reaction (ADR), and serious adverse events (SAEs) was monitored. No ADRs and SAEs related to stem cell treatment occurred during the 6-month follow-up. In terms of efficacy, the primary endpoint was increase in total walking distance (TWD). The secondary endpoint was improvement in rest pain, increase in pain-free walking distance (PFWD), toe-brachial pressure index (TBPI), transcutaneous oxygen pressure (TcPO2), and arterial brachial pressure index (ABPI). ADSCs demonstrated significant functional improvement results including increased TWD, PFWD, and rest pain reduction. No amputations were reported during the 6-month clinical trial period and in the follow-up questionnaire survey more than 2 years after the ADSC injection. In conclusion, intramuscular injection of ADSCs is very safe and is shown to prompt functional improvement in patients with severe Buerger's disease at a dosage of 300 million cells per 60 kg of body weight. However, the confirmatory therapeutic efficacy and angiogenesis need further study.

Pro-apoptotic and Growth-inhibitory Effect of IFN-ß-Overexpressing Canine Adipose Tissue-derived Mesenchymal Stem Cells Against Melanoma Cells.

BACKGROUND: Canine melanoma is the most common type of tumor in dogs. We investigated the effects of canine interferon-beta (cIFN-ß)-overexpressing adipose tissue-derived mesenchymal stem cells (cATMSCs) on apoptosis and proliferation of canine melanoma cells. MATERIALS AND METHODS: Expression of IFN-ß in cATMSCs was confirmed using reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assays. Flow cytometry was performed for cell-cycle analysis and apoptotic cell quantification of LMeC (melanoma) cells. Protein expression of cyclin D1, procaspase-3, activated caspase-3, and Bcl-2 homologous antagonist killer (Bak) was evaluated by western blot analysis. RESULTS: Decreased proportions of cells in S- and G0/G1 phases were observed in parallel with decreased cyclin D1 expression in LMeC cells treated with cIFN-ß-cATMSC-conditioned media. Protein expression of active forms of caspase 3 and Bak increased in response to treatment with cIFN-ß-cATMSC-conditioned media. CONCLUSION: IFN-ß overexpression by cATMSCs was associated with pro-apoptotic and growth-inhibitory effects on canine melanoma cells. The antitumor effects of these cells have therapeutic potential for the treatment of canine melanoma.

Health Span-Extending Activity of Human Amniotic Membrane- and Adipose Tissue-Derived Stem Cells in F344 Rats.

UNLABELLED: Aging brings about the progressive decline in cognitive function and physical activity, along with losses of stem cell population and function. Although transplantation of muscle-derived stem/progenitor cells extended the health span and life span of progeria mice, such effects in normal animals were not confirmed. Human amniotic membrane-derived mesenchymal stem cells (AMMSCs) or adipose tissue-derived mesenchymal stem cells (ADMSCs) (1×10(6) cells per rat) were intravenously transplanted to 10-month-old male F344 rats once a month throughout their lives. Transplantation of AMMSCs and ADMSCs improved cognitive and physical functions of naturally aging rats, extending life span by 23.4% and 31.3%, respectively. The stem cell therapy increased the concentration of acetylcholine and recovered neurotrophic factors in the brain and muscles, leading to restoration of microtubule-associated protein 2, cholinergic and dopaminergic nervous systems, microvessels, muscle mass, and antioxidative capacity. The results indicate that repeated transplantation of AMMSCs and ADMSCs elongate both health span and life span, which could be a starting point for antiaging or rejuvenation effects of allogeneic or autologous stem cells with minimum immune rejection. SIGNIFICANCE: This study demonstrates that repeated treatment with stem cells in normal animals has antiaging potential, extending health span and life span. Because antiaging and prolonged life span are issues currently of interest, these results are significant for readers and investigators.

Adenovirus-mediated expression of human sodium-iodide symporter gene permits in vivo tracking of adipose tissue-derived stem cells in a canine myocardial infarction model.

INTRODUCTION: In vivo tracking of the transplanted stem cells is important in pre-clinical research of stem cell therapy for myocardial infarction. We examined the feasibility of adenovirus-mediated sodium iodide symporter (NIS) gene to cell tracking imaging of transplanted stem cells in a canine infarcted myocardium by clinical single photon emission computed tomography (SPECT). METHODS: Beagle dogs were injected intramyocardially with NIS-expressing adenovirus-transfected canine stem cells (Ad-hNIS-canine ADSCs) a week after myocardial infarction (MI) development. (99m)Tc-methoxyisobutylisonitrile ((99m)Tc-MIBI) and (99m)Tc-pertechnetate ((99m)TcO4(-)) SPECT imaging were performed for assessment of infarcted myocardium and viable stem cell tracking. Transthoracic echocardiography was performed to monitor any functional cardiac changes. RESULTS: Left ventricular ejection fraction (LVEF) was decreased after LAD ligation. There was no significant difference in EF between the groups with the stem cell or saline injection. (125)I uptake was higher in Ad-hNIS-canine ADSCs than in non-transfected ADSCs. Cell proliferation and differentiation were not affected by hNIS-carrying adenovirus transfection. (99m)Tc-MIBI myocardial SPECT imaging showed decreased radiotracer uptake in the infarcted apex and mid-anterolateral regions. Ad-hNIS-canine ADSCs were identified as a region of focally increased (99m)TcO4(-) uptake at the lateral wall and around the apex of the left ventricle, peaked at 2 days and was observed until day 9. CONCLUSIONS: Combination of adenovirus-mediated NIS gene transfection and clinical nuclear imaging modalities enables to trace the fate of transplanted stem cells in infarcted myocardium for translational in vivo cell tracking study for prolonged duration.

Enhanced hepatogenic transdifferentiation of human adipose tissue mesenchymal stem cells by gene engineering with Oct4 and Sox2.

Adipose tissue mesenchymal stem cells (ATMSCs) represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of liver regeneration medicine. ATMSCs overexpressing Oct4 and Sox2 (Oct4/Sox2-ATMSCs) showed enhanced proliferation and multipotency. Hence, we hypothesized that Oct4 and Sox2 can increase "transdifferentiation" of ATMSCs into cells of the hepatic lineage. In this study, we generated Oct4- and Sox2-overexpressing human ATMSCs by liposomal transfection. We confirmed the expression of mesenchymal stem cell surface markers without morphological alterations in both red-fluorescent protein (RFP) (control)- and Oct4/Sox2-ATMSCs by flow cytometry. After induction of differentiation into hepatocyte-like cells, the morphology of ATMSCs changed and they began to appear as round or polygonal epithelioid cells. Hepatic markers were evaluated by reverse transcription-polymerase chain reaction and confirmed by immunofluorescence. The results showed that albumin was strongly expressed in hepatogenic differentiated Oct4/Sox2-ATMSCs, whereas the expression level of α-fetoprotein was lower than that of RFP-ATMSCs. The functionality of hepatocytes was evaluated by periodic acid-Schiff (PAS) staining and urea assays. The number of PAS-positive cells was significantly higher and urea production was significantly higher in Oct4/Sox2-ATMSCs compared to that in RFP-ATMSCs. Taken together, the hepatocyte-like cells derived from Oct4/Sox2-ATMSCs were mature hepatocytes, possibly functional hepatocytes with enhanced capacity to store glycogen and produce urea. In this study, we demonstrated the enhanced transdifferentiation of Oct4- and Sox2-overexpressing ATMSCs into hepatocyte-like cells that have enhanced hepatocyte-specific functions. Therefore, we expect that Oct4/Sox2-ATMSCs may become a very useful source for hepatocyte regeneration or liver cell transplantation.

Human adipose tissue-derived mesenchymal stem cells inhibit melanoma growth in vitro and in vivo.

BACKGROUND/AIM: The effects of adipose tissue-derived mesenchymal stem cells (AT-MSCs) on the growth of human malignancies, including melanoma, are controversial and the underlying mechanisms are not yet-well understood. The aim of the present study was to investigate the in vitro and in vivo anti-tumor effects of human AT-MSCs on human melanoma. MATERIALS AND METHODS: The inhibitory effect of AT-MSC-conditioned medium (AT-MSC-CM) on the growth of A375SM and A375P (human melanoma) cells was evaluated using a cell viability assay. Cell-cycle arrest and apoptosis in melanoma cells were investigated by flow cytometry and western blot analysis. To evaluate the in vivo anti-tumor effect of AT-MSCs, CM-DiI-labeled AT-MSCs were circumtumorally injected in tumor-bearing athymic mice and tumor size was measured. RESULTS: AT-MSC-CM inhibited melanoma growth by altering cell-cycle distribution and inducing apoptosis in vitro. AT-MSCs suppressed tumor growth in tumor-bearing athymic mice and fluorescence analysis showed that AT-MSCs migrated efficiently to tumor tissues. CONCLUSION: AT-MSCs inhibit the growth of melanoma suggesting promise as a novel therapeutic agent for melanoma.

Epithelial-to-Mesenchymal Transition Enhances the Cardioprotective Capacity of Human Amniotic Epithelial Cells.

The amniotic epithelium consists of cells exhibiting mature epithelial cell characteristics, but also varying degrees of stemness. We tested the hypothesis that induction of epithelial-to-mesenchymal transition (EMT) in amniotic epithelial cells (AECs) derived from human placenta enhances their capacity to support the ischemic myocardium. In response to incubation with transforming growth factor-ß1 (TGF-ß1) protein, AECs lost their cobblestone morphology and acquired a fibroblastoid shape, associated with downregulation of E-cadherin, upregulation of N-cadherin, Akt phosphorylation, and intracellular periostin translocation. EMT-AECs displayed greatly enhanced mobility and secreted gelatinase activity compared with naive AECs. The surface presentation of CD105 and CD73 decreased, and RNA microarray analysis mirrored the loss of epithelial characteristics and transcriptional profile. Unmodified AECs and EMT-AECs were then injected intramyocardially in fully immunocompetent mice after permanent LAD ligation, and heart function was followed by MRI as well as 2D speckle tracking echocardiography after 4 weeks. EMT-AEC-treated infarct hearts displayed better global systolic function and improved longitudinal strain rate in the area of interest. Although no signals of human cells were detectable by histology, infarct size was smaller in EMT-AEC-treated hearts, associated with fewer TUNEL-positive cells and upregulation of periostin, while blood vessel density was increased in both ACE- and EMT-AEC-treated hearts. We conclude that EMT enhances the cardioprotective effects of human AECs.

Survival of skin graft between transgenic cloned dogs and non-transgenic cloned dogs.

Whereas it has been assumed that genetically modified tissues or cells derived from somatic cell nuclear transfer (SCNT) should be accepted by a host of the same species, their immune compatibility has not been extensively explored. To identify acceptance of SCNT-derived cells or tissues, skin grafts were performed between cloned dogs that were identical except for their mitochondrial DNA (mtDNA) haplotypes and foreign gene. We showed here that differences in mtDNA haplotypes and genetic modification did not elicit immune responses in these dogs: 1) skin tissues from genetically-modified cloned dogs were successfully transplanted into genetically-modified cloned dogs with different mtDNA haplotype under three successive grafts over 63 days; and 2) non-transgenic cloned tissues were accepted into transgenic cloned syngeneic recipients with different mtDNA haplotypes and vice versa under two successive grafts over 63 days. In addition, expression of the inserted gene was maintained, being functional without eliciting graft rejection. In conclusion, these results show that transplanting genetically-modified tissues into normal, syngeneic or genetically-modified recipient dogs with different mtDNA haplotypes do not elicit skin graft rejection or affect expression of the inserted gene. Therefore, therapeutically valuable tissue derived from SCNT with genetic modification might be used safely in clinical applications for patients with diseased tissues.

Analysis of cell growth and gene expression of porcine adipose tissue-derived mesenchymal stem cells as nuclear donor cell.

In several laboratory animals and humans, adipose tissue-derived mesenchymal stem cells (ASC) are of considerable interest because they are easy to harvest and can generate a huge proliferation of cells from a small quantity of fat. In this study, we investigated: (i) the expression patterns of reprogramming-related genes in porcine ASC; and (ii) whether ASC can be a suitable donor cell type for generating cloned pigs. For these experiments, ASC, adult skin fibroblasts (AF) and fetal fibroblasts (FF) were derived from a 4-year-old female miniature pig. The ASC expressed cell-surface markers characteristic of stem cells, and underwent in vitro differentiation when exposed to specific differentiation-inducing conditions. Expression of DNA methyltransferase (DNMT)1 in ASC was similar to that in AF, but the highest expression of the DNMT3B gene was observed in ASC. The expression of OCT4 was significantly higher in FF and ASC than in AF (P < 0.05), and SOX2 showed significantly higher expression in ASC than in the other two cell types (P < 0.05). After somatic cell nuclear transfer (SCNT), the development rate of cloned embryos derived from ASC was comparable to the development of those derived using FF. Total cell numbers of blastocysts derived using ASC and FF were significantly higher than in embryos made with AF. The results demonstrated that ASC used for SCNT have a potential comparable to those of AF and FF in terms of embryo in vitro development and blastocyst formation.

Human adipose tissue-derived mesenchymal stem cells inhibit T-cell lymphoma growth in vitro and in vivo.

BACKGROUND/AIM: Human mesenchymal stem cells (hMSCs) are thought to be one of the most reliable stem cell sources for a variety of cell therapies. This study investigated the anti-tumor effect of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) on EL4 murine T-cell lymphoma in vitro and in vivo. MATERIALS AND METHODS: The growth-inhibitory effect of hAT-MSCs on EL4 tumor cells was evaluated using a WST-1 cell proliferation assay. Cell-cycle arrest and apoptosis were investigated by flow cytometry and western blot. To evaluate an anti-tumor effect of hAT-MSCs on T-cell lymphoma in vivo, CM-DiI-labeled hAT-MSCs were circumtumorally injected in tumor-bearing nude mice, and tumor size was measured. RESULTS: hAT-MSCs inhibited T-cell lymphoma growth by altering cell-cycle progression and inducing apoptosis in vitro. hAT-MSCs inhibited tumor growth in tumor-bearing nude mice and prolonged survival time. Immunofluorescence analysis showed that hAT-MSCs migrated to tumor sites. CONCLUSION: hAT-MSCs suppress the growth of T-cell lymphoma, suggesting a therapeutic option for T-cell lymphoma.

Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are attractive candidates for clinical repair or regeneration of damaged tissues. Oct4 and Sox2, which are essential transcription factors for pluripotency and self-renewal, are naturally expressed in MSCs at low levels in early passages, and their levels gradually decrease as the passage number increases. Therefore, to improve MSC proliferation and stemness, we introduced human Oct4 and Sox2 for conferring higher expansion and differentiation capabilities. The Oct4-IRES-Sox2 vector was transfected into human adipose tissue MSCs (ATMSCs) by liposomal transfection and used directly. Oct4 and Sox2 were successfully transfected into ATMSCs, and we confirmed maintenance of MSC surface markers without alterations in both red fluorescent protein (RFP) (control) and Oct4/Sox2-ATMSCs. Enhanced proliferative activity of Oct4/Sox2-ATMSCs was shown by WST-1 assay, and this result was further confirmed by cell counting using trypan blue exclusion for a long period. In addition, FACs cell cycle analysis showed that there was a reduction in the fraction of Oct4/Sox2-ATMSCs in G1 with a concomitant increase in the fraction of cells in S, compared with RFP-ATMSCs. Increased levels of cyclin D1 were also seen in Oct4/Sox2-ATMSCs, indicating acceleration in the transition of cells from G1 to S phase. Furthermore, Oct4/Sox2-overexpressing ATMSCs showed higher differentiation abilities for adipocytes or osteoblasts than controls. The markers of adipogenic or osteogenic differentiation were also upregulated by Oct4/Sox2 overexpression. The improvement in cell proliferation and differentiation using Oct4/Sox2 expression in ATMSCs may be a useful method for expanding the population and increasing the stemness of ATMSCs.