Extensive characterization of feline intra-abdominal adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) isolated from various tissues have been well characterized for therapeutic application to clinical diseases. However, in contrast to MSCs from other animal species, the characteristics of feline MSCs have not been fully documented. In this study, we conducted extensive characterization of feline adipose tissue-derived MSCs (fAD-MSCs). Study fAD-MSCs were individually isolated from the intra-abdominal adipose tissues of six felines. The expression levels of cell surface markers and pluripotent markers were evaluated. Next, proliferation capacity was analyzed by performing cumulative population doubling level (CPDL) and doubling time (DT) calculation assays. Differentiation potentials of fAD-MSCs into mesodermal cell lineages were analyzed by examining specific staining and molecular markers. All fAD-MSCs positively expressed cell surface markers such as CD29, CD44, CD90, CD105, CD166, and MHC-I, while CD14, CD34, CD45, and CD73 were negatively expressed. The CPDL of the fAD-MSCs was maintained until passage 5 to 6 (P5 to P6), whereas DT increased after P3 to P4. Also, stem cell-specific pluripotent markers (Oct3/4, Nanog, and SSEA-4) were detected. Importantly, all fAD-MSCs demonstrated mesodermal differentiation capacity. These results suggest that fully characterized fAD-MSCs could be beneficial when considering the use of these cells in feline disease research.

Isolation, Culturing, Characterization and Aging of Adipose Tissue-derived Mesenchymal Stem Cells: A Brief Overview

ABSTRACT The aim of this review was to describe the current state-of-the-art regarding isolation, characterization and aging of adipose tissue-derived mesenchymal stem cells (ADSCs). Mesenchymal stem cells (MSCs) have recently received widespread attention because of their potential use in tissue-engineering applications. Various studies have indicated that MSCs with a fibroblast-like morphology migrate to the sites of injury and help to regenerate damaged tissue. Over the past few years, it has been recognized that fat is not only an energy supply, but also a rich source of multipotent stem cells that can be easily harvested, isolated and selected as compared with other tissues. ADSCs are particularly interesting because of their rapid proliferation and multidirectional differentiation potential.

Effect of serum-derived albumin scaffold and canine adipose tissue-derived mesenchymal stem cells on osteogenesis in canine segmental bone defect model

Composite biological and synthetic grafts with progenitor cells offer an alternative approach to auto- or allografts for fracture repair. This study was conducted to evaluate osteogenesis of autologous serum-derived albumin (ASA) scaffolds seeded with canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) in a canine segmental bone defect model. ASA scaffold was prepared with canine serum using cross-linking and freeze-drying procedures. Beta-tricalcium phosphate (beta-TCP) was mixed at the cross-linking stage. Ad-MSCs were seeded into the scaffold and incubated for one day before implantation. After 16 weeks, the grafts were harvested for histological analysis. The dogs were divided into five groups: control, ASA scaffolds with and without Ad-MSCs, and ASA scaffolds including beta-TCP with and without Ad-MSCs. ASA scaffolds with Ad-MSCs had a significantly larger area of increased opacity at the proximal and distal host cortex-implant interfaces in radiographs 16 weeks after implantation compared to the groups with beta-TCP (p < 0.05). Histomorphometric analysis showed that ASA scaffolds with Ad-MSCs had significantly greater new bone formation than other groups (p < 0.05). These results suggest that Ad-MSCs seeded into ASA scaffolds enhanced osteogenesis in the bone defect model, but that beta-TCP in the ASA scaffold might prevent penetration of the cells required for bone healing.

Concise Review: Differentiation of Human Adult Stem Cells Into Hepatocyte-like Cells In vitro

Adult stem cells (ASCs) are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues, which are the powerful sources for cell therapy and tissue engineering. Bone marrow-derived mesenchymal stem cells (BMSCs), adipose tissue-derived mesenchymal stem cells (ADSCs), and peripheral blood monocytes (PBMCs) are the common ASCs, and many studies indicated that ASCs isolated from various adult tissues could be induced to hepatocyte-like cells in vitro. However, the isolation, culture protocols, characterization of ASCs and hepatocyte-like cells are different. This review aims to describe the isolation and culture procedures for ASCs, to summarize the molecular characterization of ASCs, to characterize function of hepatocyte-like cells, and to discuss the future role of ASCs in cell therapy and tissue engineering.

Influence of adipose tissue derived mesenchymal stem cells in combination with injectable bone substitute on osteoclastogenesis in osteoporotic rats.

The present study was designed to evaluate the influence of adipose tissue derived mesenchymal stem cell (ASCs) with or without calcium phosphate composite on osteoclastogenesis in osteoporotic rats. Mesenchymal stem cells (MSCs) were harvested from adipose tissue of both the omentum and the inguinal fat pad of male rats, as the sex mismatch, to track the MSCs fate and to ensure their homing to the injured females' femurs. The isolated ASCs were characterized via the morphological appearance, multilineage potential and the PCR detection of CD29, CD44, CD106, CD14, CD34 and CD45 surface markers. Fifty adult female albino rats were enrolled in the current study. The rats were classified into five groups: group 1 was the gonad intact control, group 2 served as untreated ovariectomized (OVX) rats, group 3 was OVX rats treated with ASCs, group 4 was OVX rats treated with ASCs with injectable bone substitute (IBS) and group 5 was OVX rats treated with IBS. The serum levels of osteoprotegerin (OPG) and receptor activator of NF-қβ ligand (RANKL) were assayed using ELISA procedure. In addition, nuclear factor-κβ (NF-κβ) gene expression level was estimated in femur bones using real time –PCR. The isolated ASCs proved their MSCs identity via their morphological appearance and multilineage potential. In addition, the isolated ASCs showed positive expression for CD29, CD45, CD44 as well as CD106 and negative expression for CD34 and CD14. Besides, the positive expression of the Y-chromosome (sry) gene detected in the ASCs treated groups indicated that the systemically delivered single dose of undifferentiated ASCs was able to home at the females' femur bones. Adipose tissue derived mesenchymal stem cells (ASCs) injection with or without calcium phosphate composite in OVX rats reversed the effect of ovariectomy on the studied biomarkers causing significant increase in serum OPG level accompanied with significant decrease in serum RANKL level. Also, significant down regulation of NF-κβ gene expression in femur bones was detected in the treated groups compared with untreated OVX group. These results clarified the good influence of ASCs against osteoclastogenesis. In addition the combination of ASCs injection with osteoinductive material injectable calcium phosphate composite (IBS), may be useful to achieve the significant antiosteoporotic effects.

Establishment of Efficacy and Safety Assessment of Human Adipose Tissue-Derived Mesenchymal Stem Cells (hATMSCs) in a Nude Rat Femoral Segmental Defect Model

Human adipose tissue-derived mesenchymal stem cell (hATMSC) have emerged as a potentially powerful tool for bone repair, but an appropriate evaluation system has not been established. The purpose of this study was to establish a preclinical assessment system to evaluate the efficacy and safety of cell therapies in a nude rat bone defect model. Segmental defects (5 mm) were created in the femoral diaphyses and transplanted with cell media (control), hydroxyapatite/tricalcium phosphate scaffolds (HA/TCP, Group I), hATMSCs (Group II), or three cell-loading density of hATMSC-loaded HA/TCP (Group III-V). Healing response was evaluated by serial radiography, micro-computed tomography and histology at 16 weeks. To address safety-concerns, we conducted a GLP-compliant toxicity study. Scanning electron microscopy studies showed that hATMSCs filled the pores/surfaces of scaffolds in a cell-loading density-dependent manner. We detected significant increases in bone formation in the hATMSC-loaded HA/TCP groups compared with other groups. The amount of new bone formation increased with increases in loaded cell number. In a toxicity study, no significant hATMSC-related changes were found in body weights, clinical signs, hematological/biochemical values, organ weights, or histopathological findings. In conclusion, hATMSCs loaded on HA/TCP enhance the repair of bone defects and was found to be safe under our preclinical efficacy/safety hybrid assessment system.

The Extracellular Matrix Affected Proliferation and Cell Adhesion of Human Adipose Tissue Derived Mesenchymal Stem Cells in vitro

PURPOSE: Human mesenchymal stem cells (hMSCs) have the potency for self-renewal and differentiation into various kinds of cells. The hMSCs are obtained from the various tissues, including adipose tissue, bone marrow and cord blood. The extracellular matrix (ECM) is an important factor that affects cell adherence, growth, migration, apoptosis and differentiation both in vitro and vivo. The adipose-derived mesenchymal stem cells (AD-MSCs) have CD29 (integrin) on the cell surface, which is the receptor for fibronectin. The aim of this study is to validate the efficacy of ECM, and especially fibronectin, for cell expansion. METHODS: The AD-MSCs were obtained from the abdominal fat of humans. These cells were seeded onto culture plates coated with fibronectin-Human (FN) and plates without ECM (control). The cells were incubated for 3 passages and the cellular morphology was simultaneously observed with microscopy. CCK-8 assay was performed to compare the proliferation ability in each condition at the same passage. Immunocytochemistry staining for integrin-beta1 was performed to observe the cell to cell interaction. RESULTS: The hAD-MSCs in the FN-coated and non-coated plates exhibited cytoplasm staining for integrin-beta1. In all the cultures, extended fibroblastic-shaped cells that turned into rhomboid cells were most frequently observed. The cell growth rates for the non coated culture plate were lower than those for the FN coated plates. After 72 hour culture under the different coated concentrations of FN and the non coated condition (control), the control group had a lower growth rate. In the culture with a FN coated plate, a significant change was observed as compared with that of the control group. We observed an increase in cell proliferation, with a maximum of 140%, on the FN coated plate by performing CCK-8 assay. In comparison, integrin beta1 on the cells was more expressed in the FN-coated plates than that in the non-coated plates. CONCLUSION: The cell morphology can be changed faster in the FN coated culture plates than that in the non coated culture plates. Because proliferation and adhesion with FN can enhance the expansion, the culture within a FN coated plate is needed to encourage hAD-MSCs to proliferate in vitro.

The Extracellular Matrix Affected Proliferation and Cell Adhesion of Human Adipose Tissue Derived Mesenchymal Stem Cells in vitro

PURPOSE: Human mesenchymal stem cells (hMSCs) have the potency for self-renewal and differentiation into various kinds of cells. The hMSCs are obtained from the various tissues, including adipose tissue, bone marrow and cord blood. The extracellular matrix (ECM) is an important factor that affects cell adherence, growth, migration, apoptosis and differentiation both in vitro and vivo. The adipose-derived mesenchymal stem cells (AD-MSCs) have CD29 (integrin) on the cell surface, which is the receptor for fibronectin. The aim of this study is to validate the efficacy of ECM, and especially fibronectin, for cell expansion. METHODS: The AD-MSCs were obtained from the abdominal fat of humans. These cells were seeded onto culture plates coated with fibronectin-Human (FN) and plates without ECM (control). The cells were incubated for 3 passages and the cellular morphology was simultaneously observed with microscopy. CCK-8 assay was performed to compare the proliferation ability in each condition at the same passage. Immunocytochemistry staining for integrin-beta1 was performed to observe the cell to cell interaction. RESULTS: The hAD-MSCs in the FN-coated and non-coated plates exhibited cytoplasm staining for integrin-beta1. In all the cultures, extended fibroblastic-shaped cells that turned into rhomboid cells were most frequently observed. The cell growth rates for the non coated culture plate were lower than those for the FN coated plates. After 72 hour culture under the different coated concentrations of FN and the non coated condition (control), the control group had a lower growth rate. In the culture with a FN coated plate, a significant change was observed as compared with that of the control group. We observed an increase in cell proliferation, with a maximum of 140%, on the FN coated plate by performing CCK-8 assay. In comparison, integrin beta1 on the cells was more expressed in the FN-coated plates than that in the non-coated plates. CONCLUSION: The cell morphology can be changed faster in the FN coated culture plates than that in the non coated culture plates. Because proliferation and adhesion with FN can enhance the expansion, the culture within a FN coated plate is needed to encourage hAD-MSCs to proliferate in vitro.

Effect of Adipose Tissue-Derived Mesenchymal Stem Cells Transplanted by Different Methods on Cardiac Function of Rabbits with Dilated Cardiomyopathy / 中国康复理论与实践

@#Objective To investigate the effect of adipose tissue-derived mesenchymal stem cells (ADMSCs) transplanted by different Methods on cardiac function of rabbits with dilated cardiomyopathy.Methods 50 white ears rabbits were given doxorubicin by intraperitoneal injection to induce dilated cardiomyopathy, and randomly divided into the model group 1, model group 2 and control group. The ADMSCs isolated and cultured in vitro were transplanted into dilated cardiomyopathy rabbits of model group 1 through multi-points injection in myocardium, and were transplanted into rabbits of model group 2 through coronary artery transplant. Those in the control group were treated with IMEM medium of the same volume through multi-points injection in myocardium. Rabbits were fed for 4 week successively and then were killed to obtain heart sample, and the survival and differentiation of transplanted cells were observed through fluorescence microscope. Before transplantation and 4 weeks after transplantation, all rabbits received ultrasonic cardiogram test and haemodynamics test to determine cardiac function.Results After transplant through the two different Methods , ADMSCs could survive and differentiate in myocardial cells. 4 weeks after transplantation, the left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) reduced more significantly in the model group 2 compared with the model group 1 ( P<0.05～0.01), and LVSV, ejection fraction (EF), left ventricular systolic pressure, +dp/dtmax, -dp/dtmax increased more significantly ( P<0.05～0.01).Conclusion Transplanted ADMSCs can survive and differentiate in myocardial cells, and improve the heart function in rabbits with dilated cardiomyopathy. It is more conducive to the effectiveness of ADMSCs to improve the heart function through the method of coronary artery transplant.

The Effects of Adipose Tissue-Derived Mesenchymal Stem Cells on the Formation ofEpidermis and Basement Membrane in Artificial Skin Models / 대한피부과학회지

BACKGROUND: There is an increasing need for making a more ideal artificial skin model. OBJECTIVE: To evaluate the effects of adipose tissue-derived mesenchymal stem cells (ATMSC) on the formation of epidermis and basement membrane in artificial skin models. METHODS: ATMSC were isolated from lipo-aspirated fat tissues, and their phenotypes were confirmed by cell surface markers. Three kinds of artificial skin models were made using three different dermal substitutes. The dermal substitutes in the three models contained fibroblasts only, fibroblasts together with ATMSC or ATMSC only. The formation of epidermis and basement membrane was evaluated by immunohistochemical stains and transmission electron microscopy. RESULTS: Among the three models, the model with both fibroblasts and ATMSC in the dermal substitute showed the most excellent formation of epidermis and, especially, basement membrane. In this model, the basement membrane proteins, laminin and type IV collagen, were expressed most apparently at the dermo-epidermal junction and, lamina lucida, lamina densa and anchoring fibrils were most evidently observed under transmission electron microscopy. Whereas, the model with only ATMSC did not show keratin 1 expression, suggesting that the 'skin-type' stratified squamous epithelium was not formed well. CONCLUSION: ATMSC together with fibroblasts can be used effectively in constructing artificial skin models.

The Effect of Human Adipose Tissue Derived Mesenchymal Stem Cells and Growth Hormone on the Recovery of Neurological Deficits due to Experimental Spinal Cord Injury in Rat

PURPOSE: Human adipose tissue-derived mesenchymal stem cells(hATSCs) can be differentiated into multiple mesenchymal lineages, including bone, cartilage, and muscle. And growth hormone play important roles in the normal growth and development of the CNS. In this study, we explored whether the transplanted hATSCs and growth hormones could improve functional recoveries from rats with contusive spinal cord injury. METHODS: We divided 30 female rats, which were subjected to a weight driven implant spinal cord injury, into 3 groups with 10 rats each; Group A as a control group, group B with hATSCs transplantation on injured region, and group C with hATSCs transplantation and GH administration for 7 days. Then, we researched their neurologic functional recoveries before and 2, 4, and 8 weeks after transplantation using Basso-Beattie-Bresnahan (BBB) locomotor rating scale. And we checked Y- chromosome positive cells by FISH(Fluorescent in situ hybridization) to identify the survival of transplanted mesenchymal stem cells. RESULTS: After 4 weeks of transplantation, the group B and group C showed significant improvement of neurologic function on BBB locomotor rating scale in comparison with the group A(Group A: 13.1+/-0.58, Group B: 14.6+/-0.69, Group C: 14.9+/-0.56). Moreover, the group C displayed meaningful recovery of neurologic function after 8 weeks in comparison with group B (Group B: 15.7+/-0.63, Group C: 16.5+/-1.14). The group A, the control one, improved for 5 weeks after injury, and had no more recovery. On the other hand, Group B and C showed the improvement of neurologic function continuously for 9 weeks after injury. CONCLUSION: In this study, we found out that hATSCs transplantation have an effect on neurologic functional recovery of spinal cord injured rat and GH injection seems to bring the synergistic results on this good tendency.

Inducing Mesenchymal Stem Cells from Adult Human Adipose Tissue to Differentiate into Cardiomyocytes In Vitro / 中国康复理论与实践

@# ObjectiveTo study the differentiation of adipose tissue-derived mesenchymal stem cells (ADMSCs) into cardiomyocytes in vitro. MethodsADMSCs were isolated and purified by the method of digesting and adhering to the culture plastis. The third generation of cells were determined with immunocytochemistry method and induced by 5-aza. On the 7th, 14th, 21st, 28th day after being induced, the surface antigen of myoblast cell and cardiac myocyte were determined, the expression of gene of GATA4 and Nkx2.5 were tested, and the content of atrial natriuretic polypeptide (ANP) assayed. ResultsImmunocytochemical staining showed CD44, CD13, CD105 positive, CD45, CD34, HLA-DR, factor Ⅷ negative. After being induced by 5-aza, the direction of cell arraying was gradually similar. The cells were stained positively for Desmin, α-Sarcomeric Actin, myosin heavy chain and Troponin T. The cells express GATA4 and Nkx2.5 and secrete ANP. ConclusionThere was mesenchymal stem cells in human adipose tissue. 5-aza may induce ADMSCs to differentiate into cardiomyocytes in vitro.