BACKGROUND AND OBJECTIVES: Hydroxyapatite has biocompatibility and bioactivity and similar to bone of in human body. The purpose of this study is to evaluate osteogenic differentiation of bone marrow stem cell (BMSC) in PLGA Scaffold added various ratio of hydroxyapatite (HAp). METHODS AND RESULTS: PLGA and PLGA/HAp scaffold were prepared using solvent casting/salt-leaching method. BMSC was seeded on the PLGA and PLGA/HAp scaffold and the samples were cultured in 37degrees C incubator with 5% CO2 for 28 days. Alkaline phosphatase (ALP) was carried out to evaluate alkaline phosphatase activity at 1, 3, 7, 10 and 14 days. Alizarin Red S stating was performed to identify calcium in scaffold at 1, 7, 14, 21 and 28 days. Compressive strength was measured to evaluate mechanical property of scaffold. To confirm cell viability, MTT was carried out at 1, 3, 7, 14 and 28 days. RT-PCR was performed to verify specific marker expression of osteoblast and stem cell at 7, 14, 21 and 28 days. CONCLUSIONS: Osteogenic differentiation of BMSC was confirmed through ALP, RT-PCR, and alizarin red S staining in this study. These results suggest that HAp helps osteogenic differentiation of BMSC.
Alkaline Phosphatase ; Anthraquinones ; Bone Marrow ; Calcium ; Cell Survival ; Compressive Strength ; Durapatite ; Human Body ; Incubators ; Lactic Acid ; Osteoblasts ; Polyglycolic Acid ; Seeds ; Stem Cells

BACKGROUND AND OBJECTIVES: Acute kidney injury (AKI) represents a major clinical problem with high mortality and limited treatment protocols. This study was planned to evaluate the therapeutic effectiveness of bone marrow-derived mesenchymal stem cells (BM-MSCs) in a rat model of ischemia/reperfusion (I/R) AKI. METHODS AND RESULTS: This study was carried out on thirty adult male albino rats. Animals were divided equally into three groups. Group I (control sham-operated group) (n=10), were subdivided equally into two subgroups; Ia and Ib. The experimental group (n=20) were all subjected to I/R injury by clamping both renal pedicles for 40 minutes. Half of the I/R animals did not receive MSC therapy (group II) [non-MSC treated group]. The other half of the I/R animals received single intravenous injection of PKH26 labelled BM-MSCs immediately after removal of the clamps and visual confirmation of reflow (group III) [MSC treated group]. Animals were sacrificed 24 hrs (subgroups IIa & IIIa) and 72 hrs (subgroups IIb & IIIb) after intervention. Serological measurements included serum urea and creatinine. Kidney specimens were processed for H&E, PAS and PCNA. Mean % of renal corpuscles with affected glomeruli, mean % of affected tubules, mean area % of PAS-positive reaction and mean area % of PCNA immunoreactivity were measured by histomorphometric studies and statistically compared. MSCs-treated group exhibited protection against renal injury serologically and histologically. CONCLUSIONS: Results of the present study suggest a potential reno-protective capacity of MSCs which could be of considerable therapeutic promise for cell-based management of clinical AKI.
Acute Kidney Injury ; Adult ; Animals ; Clinical Protocols ; Constriction ; Creatinine ; Humans ; Injections, Intravenous ; Kidney ; Male ; Mesenchymal Stromal Cells ; Organic Chemicals ; Proliferating Cell Nuclear Antigen ; Rats ; Urea

BACKGROUND AND OBJECTIVES: Irradiated wound healing is a highly complex and dynamic process. The latest technology making a huge difference in this process is stem cell therapy. The goal of this study was to evaluate the use of bone marrow-derived mesenchymal stem cells (BM-MSCs) or human amniotic epithelial cells (HAECs) in the healing of irradiated wounds. METHODS AND RESULTS: Forty five male albino rats were subjected to whole body 6 gray gamma radiations. One day post irradiation, full-thickness incisional wound was created in the tibial skin. The rats were randomly equally divided into three groups. The incisions of the first group (gp I) were injected intra-dermally with saline before stitching and those of both the second (gp II) and the third groups (gp III) were intradermally injected with BM-MSCs and HAECs before stitching respectively. Animals were sacrificed after the third, seventh and fourteenth days postoperative. The healing process was assessed histopathologically. CXCL-5, SDF-1 and Transforming growth factor-beta 1 (TGF-beta1) expression were also detected in biopsies from all wounds. Expression of TGF-beta1 in gp I was more than the other groups leading to severe inflammation, deficient healed dermis and delayed reepithelialization. SDF-1 expression was high in gp II while CXCL-5 expression was high in gp III causing accelerated wound healing. BM-MSCs showed a great effect on the quality of the dermis, while superiority of the epithelium and its appendages were achieved in HAECs group. CONCLUSIONS: Using BM-MSCs and HAECs could be used safely in case of irradiated wounds.
Animals ; Biopsy ; Dermis ; Epithelial Cells ; Epithelium ; Gamma Rays ; Humans ; Inflammation ; Male ; Mesenchymal Stromal Cells ; Rats ; Skin ; Stem Cells ; Transforming Growth Factor beta1 ; Wound Healing

BACKGROUND AND OBJECTIVES: Half of patients with critical limb ischemia (CLI) are ineligible for revascularization at diagnosis. The aim of this study was to assess the safety and feasibility of intramuscular human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) therapy in patients with CLI due to atherosclerosis obliterans (ASO) or thromboangiitis obliterans (TAO). METHODS AND RESULTS: A total of eight patients (all male, median age 52 years, range 31~77) with CLI were enrolled in this phase I trial. All patients were considered ineligible for further revascularization to improve CLI. We injected 1x10(7) hUCB-MSCs per single dose intramuscularly into the affected limb. The primary end points of safety were occurrence of adverse events (procedure-related complication, allergic reaction to hUCB-MSCs, graft-versus-host disease, cardiovascular and cerebrovascular events) and improvement of symptoms/clinical parameters (healing of foot ulcer, ankle-brachial index, and pain-free walking distance). Angiogenesis was measured with conventional angiography and scored by an independent reviewer. There were four adverse events in three patients. One patient, developed whole body urticaria after injection on treatment day, which disappeared after one day of antihistamine treatment. The other adverse events included diarrhea, oral ulceration, and elevation of serum creatinine level; all conditions improved without treatment. Abnormal results of laboratory parameters were not detected in any patients. Three of four ulcerations (75%) healed completely. Angiographic scores increased in three of eight patients. CONCLUSIONS: This phase I study demonstrates that intramuscular hUCB-MSC injection is a safe and well tolerated treatment for patients with end-stage CLI due to ASO and TAO.
Angiography ; Ankle Brachial Index ; Arterial Occlusive Diseases ; Atherosclerosis ; Creatinine ; Diarrhea ; Extremities ; Fetal Blood ; Foot Ulcer ; Graft vs Host Disease ; Humans ; Hypersensitivity ; Ischemia ; Male ; Mesenchymal Stromal Cells ; Oral Ulcer ; Oxalates ; Stem Cells ; Thromboangiitis Obliterans ; Troleandomycin ; Ulcer ; Umbilical Cord ; Urticaria ; Walking

BACKGROUND AND OBJECTIVES: Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. METHODS AND RESULTS: Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA-4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. CONCLUSIONS: Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.
Animals ; Blastocyst ; Buffaloes ; Collagen ; Collagen Type I ; Drug Combinations ; Embryonic Stem Cells ; Extracellular Matrix ; Feeder Cells ; Female ; Fibroblasts ; Fibronectins ; Granulosa Cells ; Humans ; Laminin ; Mental Competency ; Models, Animal ; Oviducts ; Proteoglycans ; Stage-Specific Embryonic Antigens ; Stem Cell Research

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells have delivered new approaches to the management of wound healing in severe skin injuries. This work was planned to evaluate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on healing of induced full thickness skin wounds in albino rats using topical & systemic injections. METHODS AND RESULTS: Forty adult male albino rats were classified into 2 groups after induction of full thickness skin wound; untreated group and stem cell-treated group. The latter was further subdivided into topically and systemically treated ones. BMSCs were isolated & labeled by PKH26 before injection. Healing of wounds was evaluated grossly. Skin biopsies were obtained one & three weeks after wound induction. Sections were stained with Hematoxylin & Eosin, Masson's trichrome and immunohistochemichal stain for vascular endothelial growth factor (VEGF). Epidermal thicknesses and mean area percent of both collagen fibers & VEGF immunopositive cells were measured using image analyzer & results were subjected to statistical analysis. PKH26 fluorescent-labeled cells were found in the regenerated epidermis, hair follicles and dermis in BMSCs-treated groups. By the end of the third week, the wounds of BMSCs-treated groups showed full regeneration of epidermis, re-organization of collagen and decrease in VEGF immunopositive cells. Delayed wound healing was seen in 20% of systemically treated rats. Significant increase in the mean area percent of collagen fibers was detected in topically treated group. CONCLUSIONS: Both methods of BMSCs injection were effective in healing of full thickness skin wound but topical method was more effective.
Adult ; Animals ; Biopsy ; Bone Marrow ; Collagen ; Dermis ; Eosine Yellowish-(YS) ; Epidermis ; Hair Follicle ; Hematoxylin ; Humans ; Male ; Mesenchymal Stromal Cells ; Organic Chemicals ; Rats ; Regeneration ; Skin ; Vascular Endothelial Growth Factor A ; Wound Healing

BACKGROUND AND OBJECTIVES: The rapidly increasing number of diabetic patients across the world drew the attention to develop more effective therapeutic approaches. Recent investigations on newly differentiated insulin producing cells (IPCs) revealed that they could be derived from embryonic, adult mesenchymal and hematopoietic stem cells. This work was planned to evaluate the role of StemEnhance (Aphanizomenon flos-aquae [AFA] plant extract) in mobilizing naturally occurring bone marrow stem cells as well as in improving streptozotocin-induced diabetic rats. METHODS AND RESULTS: Twenty adult male albino rats were divided into four groups namely the control, the diabetic, the positive control-StemEnhance and the diabetic-StemEnhance groups. After diabetes induction by streptozotocin (STZ), rats received StemEnhance for four weeks. The mean number of blood CD34 immunopositive cells was measured by flowcytometry and random blood sugar was measured weekly. The pancreas was removed from the sacrificed rats and processed for staining with H&E and immunohistochemical staining for CD34+ve and insulin +ve cells. CD34+ve cells increased in the blood after introduction of StemEnhance. CD34+ve cells were observed in the pancreas and the insulin producing cells in the islets of Langerhans were increased from the second to the fourth week of treatment. Blood glucose level improved but it was still higher than the control level after four weeks of StemEnhance treatment. CONCLUSIONS: This work points to the significant role of StemEnhance in stem cell mobilization and the improvement of diabetes mellitus.
Adult ; Animals ; Blood Glucose ; Bone Marrow ; Diabetes Mellitus ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cells ; Humans ; Insulin ; Islets of Langerhans ; Male ; Pancreas ; Plants ; Rats ; Stem Cells ; Streptozocin

BACKGROUND: The normal cells derived from human embryonic stem cells (hESCs) are regarded as substitutes for damaged or dysfunctional adult cells. However, tumorigenicity of hESCs remains a major challenge in clinical application of hESC-derived cell transplantation. Previously, we generated monoclonal antibody (MAb) 57-C11 specific to the surface molecule on undifferentiated hESCs. The aim of this study is to prove whether 57-C11-positive hESCs are pluripotent and tumorigenic in immunodeficient mice. METHODS: Undifferentiated hESCs were mixed with retinoic acid (RA)-differentiated hESCs at different ratios prior to 57-C11-mediated separation. To isolate 57-C11-positive hESCs from the mixture, biotinylated 57-C11 and streptavidin-coated magnetic beads were added to the mixture. Unbound 57-C11-negative hESCs were first isolated after applying magnet to the cell mixture, and 57-C11-bound hESCs were then released from the magnetic beads. In order to measure the efficiency of separation, 57-C11-positive or -negative hESCs were counted after isolation. To evaluate the efficiency of teratoma formation in vivo, 57-C11-positive or negative cells were further injected into left and right, respectively, testes of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. RESULTS: Approximately 77~100% of undifferentiated hESCs were isolated after applying 57-C11-coated magnetic beads to the mixed cell populations. Importantly, teratomas were not observed in NOD/SCID mice after the injection of isolated 57-C11-negative hESCs, whereas teratomas were observed with 57-C11-positive hESCs. CONCLUSION: 57-C11-positive hESCs are pluripotent and tumorigenic. The combination of 57-C11 and magnetic beads will be useful to eliminate remaining undifferentiated hESCs for the safe cell transplantation.
Adult ; Animals ; Cell Transplantation ; Human Embryonic Stem Cells* ; Humans* ; Mice ; Teratoma ; Testis ; Transplants ; Tretinoin

BACKGROUND: Mesenchymal stem cells (MSCs), have been suggested as a potential choice for treatment of male infertility. Yet, the effects of MSCs on regeneration of germinal epithelium of seminiferous tubules and recovery of spermatogenesis have remained controversial. In this research, we have evaluated and compared the fate of autologous bone marrow (BM)-MSCs during three different periods of time- 4, 6 and 8 weeks after transplantation into the testes of busulfan-induced infertile male rats. METHODS: Rats BM samples were collected from tibia bone under anesthesia. The samples were directly cultured in culture medium. Isolated, characterized and purified BM-MSCs were labeled with PKH26, and transplanted into the testes of infertile rats. After 4, 6 and 8 weeks, the testes were removed and underwent histological evaluations. RESULTS: Immunohistochemical analysis showed that transplanted BM-MSCs survived in all three groups. Some of the cells homed at the germinal epithelium and expressed spermatogonia markers (Dazl and Stella). The number of homed spermatogonia-like cells in 4-week testes, was more than the 6-week testes. The 8-week testes had the least numbers of homed cells (p<0.05). Immunostaining for vimentin showed that BM-MSCs did not differentiate into the sertoli cells in the testes. CONCLUSIONS: From our results, it could be concluded that, autologous BM-MSCs could survive in the testis, migrate onto the seminiferous tubules basement membrane and differentiate into spermatogonia. Although, no more differentiation was observed in the produced spermatogonia, generation of such endogenous GCs would be a really promising achievement for treatment of male infertility using autologous stem cells.
Anesthesia ; Animals ; Basement Membrane ; Bone Marrow* ; Epithelium ; Germ Cells* ; Humans ; Infertility, Male ; Male* ; Mesenchymal Stromal Cells* ; Rats* ; Regeneration ; Seminiferous Tubules ; Sertoli Cells ; Spermatogenesis ; Spermatogonia ; Stem Cells ; Testis* ; Tibia ; Transplantation ; Vimentin

BACKGROUND AND OBJECTIVES: Mesenchymal stromal cells (MSCs) have great therapeutic potential, particularly in the process of tissue repair and immunomodulation through the secretion of biomolecules. Thus, the aim of this study was to evaluate the hypothesis that intramuscular transplantation of allogeneic MSCs obtained from equine umbilical cord (UC-MSCs) is safe, demonstrating that this is a suitable source of stem cells for therapeutic use. METHODS AND RESULTS: For this, UC-MSCs were cultured, characterized and cryopreserved for future transplantation in six healthy mares. On day 0, transplantation of three million UC-MSCs diluted in Hank’s Balanced Solution (HBSS) was performed on right and left sides of the rump muscle. As a control, HBSS injections were performed caudally in the same muscle. Muscle biopsies were obtained as a control 30 days before transplantation (D-30). The biopsies were collected again on day 2 (left side) and day 7 (right side) post transplantation and examined histologically. All procedures were preceded by ultrasound examination and blood sampling. Hematologic evaluation remained within normal limits and no differences were observed between time points (p>0.05). Ultrasound examination was suggestive of inflammation 48 hours after transplantation in both groups (control and treated). At histological evaluation it was found only discrete inflammation signals between D-30×D2 (p<0.05) in the treated group, without differences (p> 0.05) between the groups at different time points. CONCLUSIONS: Equine UC-MSCs under the experimental conditions did not promote severe inflammation that causes tissue damage or lead to its rejection by the host organism and therefore has a good potential for clinical use.
Biopsy ; Immunomodulation ; Inflammation ; Mesenchymal Stromal Cells* ; Stem Cells ; Transplantation ; Ultrasonography ; Umbilical Cord*