Clinical studies using stem cells for treatment of retinal diseases: state of the art / Estudos clínicos com uso de células-tronco para tratamento de doenças da retina: estado da arte

ABSTRACT Degenerative retinal diseases such as retinitis pigmentosa, Stargardt's macular dystrophy, and age-related macular degeneration are characterized by irreversible loss of vision due to direct or indirect photoreceptor damage. No effective treatments exist, but stem cell studies have shown promising results. Our aim with this review was to describe the types of stem cells that are under study, their effects, and the main clinical trials involving them.

RESUMO As doenças degenerativas da retina, como retinose pigmentar, distrofia macular de Stargardt e degeneração macular relaciona à idade, são caracterizadas por perda irre versível da visão devido a danos diretos ou indiretos aos fotorreceptores. Não existem tratamentos eficazes, porém os estudos com células-tronco mostraram resultados promissores. Nosso objetivo com esta revisão foi descrever os tipos de células-tronco em estudo, seus efeitos e os principais ensaios clínicos que as envolvem.

Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Ischaemic-Reperfused Hearts in Adult Rats with Established Chronic Kidney Disease

BACKGROUND AND OBJECTIVES: Bone marrow-derived mesenchymal stem cells (BM-MSCs) are adult multipotent non-haematopoietic stem cells that have regeneration potential. The current study aimed to detect the ability of BM-MSCs to improve kidney and cardiac functions in adult rats with established chronic kidney disease. METHODS: Rats were divided into sham-operated control, untreated sub totally nephrectomised and treated sub totally nephrectomised groups. Body weight, kidney and cardiac tissue weights, plasma creatinine and urea levels and arterial blood pressure were measured. ECG was recorded, and an in vitro isolated heart study was performed. Results: Stem cell treatment decreased the elevated plasma creatinine and urea levels and decreased systolic, diastolic and mean arterial blood pressure values. These changes were accompanied by a decrease in glomerular hypertrophy with apparent normal renal parenchyma. Additionally, BM-MSCs shortened Q-To and Q-Tc intervals, all time to peak tension values, the half relaxation value at 30 min of reperfusion and the contraction time at 15 and 30 min of reperfusion. Moreover, stem cell treatment significantly increased the heart rate, QRS voltage, the peak tension at the 15- and 30-min reperfusion time points and the peak tension per left ventricle at the 30-min reperfusion time point compared to the pre-ischaemia baseline. BM-MSCs resolve inter muscular oedema and lead to the re-appearance of normal cardiomyocytes. This improvement occurs with the observations of BM-MSCs in renal and heart tissues. CONCLUSIONS: BM-MSCs can attenuate chronic kidney disease progression and the associated cardiac electrophysiological and inotropic dysfunction.

Bone Marrow Stem Cells Anti-liver Fibrosis Potency: Inhibition of Hepatic Stellate Cells Activity and Extracellular Matrix Deposition

Transplantation of bone marrow derived stem cells (BMSCs) has been reported inhibits liver fibrosis. Several in vitro studies by co-culturing BMSCs and hepatic stellate cells (HSCs) indirectly or directly in 2D models showed inhibition of HSC as the key player in liver fibrosis. In this study, we investigated direct effect of BMSCs on HSCs by co-culturing BMSCs and HSCs in 3D model as it represents the liver microenvironment with intricate cell-cell and cell-matrix interactions. Primary isolated rat HSCs and BMSCs were directly co-cultured at 1:1 ratio with hanging drop method. The monoculture of rat HSCs served as positive control. Mono-culture and co-culture samples were harvested on day 3, 5 and 7 for histological analysis. The samples were analyzed for extracellular matrix deposition by Masson's Trichrome staining, tenascin-C immunocytochemistry, resting HSC's state as shown by positive Oil Red O stained cells. Our results indicated CD90+CD34− BMSCs anti-liver fibrosis potency as evidenced by higher proportion of Oil Red O-positive cells in the co-culture group compared to the monoculture group and the significant decrease in extracellular matrix deposition as well as the decrease in tenascin-C expression in the co-culture group (p<0.05) compared to the monoculture group. These findings demonstrate that BMSCs have a potential therapeutic effect against liver fibrotic process through their capacity to inhibit HSCs activation and their effect in minimizing extracellular matrix deposition.

Role of miR-122 in bone marrow derived stem cell therapy for acute liver injury in rats / 吉林大学学报(医学版)

Objective To investigatet the effects of miR-122 in the therapy of bone marrow derived stem cells (BMSCs)for acute liver injury in the rats,and to clarify the mechanism.Methods The BMSCs were isolated from the bone marrow of male rats by density gradient centrifugation.The BMSCs were divided into transfection group and control group.The BMSCs in transfection group were transfected with miR-122 mimics by liposome,while the BMSCs in control group were not.60 SD rats with acute liver injury induced by 10%CCl4 were randomly divided into control group (the saline was injected through mainline),normal treatment group (the normal BMSCs were injected through mainline) and experimental therapy group (the BMSCs transfected with miR-122 mimics by liposome were injected through mainline)(n=20).The liver function and tissue pathology were examined at 1 d, 7 d and 14 d after transplantation.Results The expression level of ALB in BMSCs was up-regulated,while the AFP expression level was down-regulated after the transfection of miR-122 mimics.At 1 d after transfection of BMSCs,the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities had no significant difference between normal treatment group and experimental therapy group.At 7 d and 14 d after transfection of BMSCs,the serum ALT and AST activities in experimental therapy group were obviously lower than those in normal treatment group (P <0.05).The liver congestion,cytoplasm degeneration and liver cell necrosis in experimental therapy group were improved compared with normal treatment group.Conclusion The up-regulation of miR-122 expression in BMSCs would promote its differentiation into hepatocyte like cells,which plays a role in promoting the recovery of liver injury.

Thinking and actuality of induced pluripotent stem cells to treat spinal cord injury / 中国组织工程研究

BACKGROUND: Currently, therapies for spinal cord injury include steroid pulse therapy, surgical decompression and stem cel therapy. A lot of work has been focused on stem cel therapy for spinal cord injury which has become a hot spot. OBJECTIVE: To summarize the research status and progress in induced pluripotent stem cells for treatment of spinal cord injury. METHODS: A computer-based online search of PubMed (1980-01/2011-11) was performed for articles in English using the keywords of “spine injury, induced pluripotent stem cells, IPS”RESULTS AND CONCLUSION: A total of 35 articles were included in result analysis, in which, stem cel therapy for treatment of spinal cord injury is agreed or supported. Induced pluripotent stem cells are isolated directly from the body, which solves ethical and moral issues in the transplantation of stem cells, meanwhile avoids al ograft rejection and also provides a large source of cells. Stem cel therapy for spinal cord injury is widely used in animal experiments but few in clinical application. However, stem cel therapy has a good effect in the animal experiments, and shows a higher safety. The existing problems of induced pluripotent stem cells to treat spinal cord injury mainly include immature differentiating method of induced pluripotent stem cells and more complications. Therefore, induced differentiation and security of induced pluripotent stem cells for treatment of spinal cord injury wil become a research key.

Tissue-engineered tendon construction using bone marrow mesenchymal stem cells induced by bone morphogenetic protein 12 / 中国组织工程研究

BACKGROUND: The previous methods for the repair of tendon defect include end-to-end method, autologous tendon graft, tendon al ograft or artificial tendon transplantation, but each method has its advantages and disadvantages. OBJECTIVE: To investigate the feasibility of constructing tissue-engineered tendon by rabbit bone marrow mesenchymal stem cells as seed cells which were induced by bone morphogenetic protein 12 and with col agen-polyglycolic acid composite as frameworks in the repair of rabbit tendon defects. METHODS: Bone marrow was separated from rabbit proximal femur to harvest cells, and the cells were passaged to the second generation and induced with 10 μg/L bone morphogenetic protein 12. Then the passage 2 cells were implanted into the prefabricated tissue-engineered tendon on the polyglycolic acid stitch with certain percentage together with col agenⅠsolution. The rabbits were used to establish the Achil es tendon defect models, and different methods were used to repair Achil es tendon defect: tissue-engineered tendon, col agenⅠ-polyglycolic acid stitch and end-to-end suturing in silk. Morphology, mechanics and shistopathology of the tissue-engineered tendon were observed. RESULTS AND CONCLUSION: Pathomorphological observation of histological section after 12 weeks showed that multiple fusiform fibroblasts were homogeneously distributed in col agen in the direction of stress mechanics.Fibrocytes increased obviously, new smal vessels could be seen and col agen was found aligned compactedly. In col agen Ⅰ-polyglycolic acid stitch group, a part of fibroplasia hyperplasia accompanied by granulation tissue formation could be seen, the col age fibers were in loose filamentous network and the cells were distributed disorderly and unevenly. Granulation tissue formed around the fibrous tissue in the silk group. Biomechanics strength in bone morphogenetic protein 12+polyglycolic acid group was better than that in the col agen Ⅰ-polyglycolic acid group, and there was significant difference when compared with suture silk group. The biomechanics strength of the bone morphogenetic protein 12+polyglycolic acid group was lower than that of normal tendon. It is possible to construct a tissue-engineered tendon with autologous bone marrow mesenchymal stem cells as seed cells induced by bone morphogenetic protein 12 and with the col agen-polyglycolic acid as the framework. Constructed tissue-engineered tendon has biomechanics characteristics and can be used to repair Achil es tendon defect.

Therapeutic potential of non-adherent bone marrow-derived stem cells for acute radiation injury / 中国组织工程研究

BACKGROUND: Effective treatment for severed acute radiation sickness (over 8 Gy) has not been obtained at present. Mesenchymal stem cells, which are shown to secrete hematopoietic cytokines and support hematopoietic progenitors, play an important role in cute radiation sickness. OBJECTIVE: To investigate the therapeutic potential of non-adherent bone marrow-derived stem cells in the treatment of acute radiation injury induced by 8.5 Gy X-ray irradiation, as wel as the mechanisms involved. METHODS: Non-adherent marrow-derived stem cells from the long bone of fetal limbs were col ected for analyzing surface antigens, cel cycle, osteogenic and adipogenic differentiation potential, and expressions of vascular endothelial growth factors and Annexin A2. After being exposed to 8.5 Gy total body irradiation, BALB/C mice were randomly assigned into transplantation group and control group. Mice in the transplantation group were given 3×106 CFDA-SE labeled human non-adherent bone marrow-derived stem cells, and those in the control group were given 0.3 mL normal saline. Then, the survival rate, peripheral white blood cells at different time, pathologic change and angiogenesis of the bone marrow were observed. RESULTS AND CONCLUSION: After X-ray irradiation, transplanted non-adherent mesenchymal stem cells appeared to have a homing to the site of injury. The survival rate of mice in the transplantation group was much higher than that in the control group. Compared with the control group, the white blood cells in the transplantation group decreased more slowly while recovered more rapid: the nadir appeared at day 14 after transplantation while it recovered within 30 days. The bone marrow of mice in the transplantation group regenerated more actively and had more hematopoietic islands than those in the control group on day 21. In addition, bone marrow angiogenesis of the transplantation group was more obvious than that of the control group. In conclusion, human fetal non-adherent bone marrow-derived stem cells could promote bone marrow angiogenesis in a mouse model of acute radiation injury, through which they could play an important role in tissue regeneration of acute radiation injury.

Construction of oxalate-degrading adult bone marrow mesenchymal stem cell lines / 中国组织工程研究

BACKGROUND: High oxalic acid urine is a risk factor for stone formation. Constructing cel lines with high oxalate metabolic ability using genetic engineering and stem cel technology wil become the effective method to prevent and treat calcium oxalate stone. OBJECTIVE: To construct the adult bone marrow mesenchymal stem cel lines that can decompose the oxalic acid, through co-transfecting the oxalic acid degradation genes Frc and Oxc of oxalobacter formigenes into the normal adult bone marrow mesenchymal stem cells. METHODS: Frc and Oxc were amplified by PCR, and the eukaryotic expression vectors of pLEGFP-N1-myc-Frc and pBaBE-puro-flag-Oxc were constructed, then co-transfected into the normal adult bone marrow mesenchymal stem cells. The non-transfected bone marrow mesenchymal stem cells and the cells transfected with empty vectors were as control. Western blot was performed to detect the expression of the objective genes;the concentration of oxalate in the culture medium after transgenic was determined by ion chromatography. RESULTS AND CONCLUSION: Restriction enzyme digestion and sequencing results showed that the Frc and Oxc genes were successful y amplified, and the vectors of pLEGFP-N1-myc-Frc and pBaBE-puro-flag-Oxc were constructed. After tranfected into the bone marrow mesenchymal stem cells, the Western blot results showed that transfected bone marrow mesenchymal stem cells could stably express the target protein myc-formyl coenzyme A transferase enzyme and the flag-oxalyl coenzyme A decarboxylase; ion chromatography test results showed with the prolonging of the culture time, the concentration of oxalic acid in the human bone marrow mesenchymal stem cel culture medium transfected with target gene was decreased gradual y. While there was no target protein expression in the non-transfected human bone marrow mesenchymal stem cells as wel as the cells transfected with empty vectors. The cells had the ability of oxalate-degradation. The experiment successful y constructs the adult bone marrow mesenchymal stem cel lines that can decompose the oxalic acid, and the cel lines have the ability of oxalate-degradation and can stably express the oxalate decomposition proteins Frc and Oxc.

Bone marrow mesenchymal stem cells from diabetic versus normal rats for treatment of myocardial infarction / 中国组织工程研究

BACKGROUND: Current studies have shown that bone marrow mesenchymal stem cells from normal or young people usual y serve as a source of transplanted cells in stem cel transplantation treatment of myocardial infarction. OBJECTIVE: To compare the therapeutic effects of bone marrow mesenchymal stem cells from diabetic and normal rats on myocardial infarction. METHODS: Under sterile conditions, bone marrow mesenchymal stem cells from normal and diabetic rats were harvested. Then, rat models of myocardial infarction were established and randomly divided into three groups:100 μL cellsuspension containing 105-106 bone marrow mesenchymal stem cells from F2 normal or diabetic rats was injected into myocardial infarction lesions, and 100 μL Dulbecco’s modified Eagle’s medium containing 20%fetal bovine serum was injected serving as blank control. After 1 month, hematoxylin-eosin staining for myocardial infarction lesions was performed for histomorphological observation. Bcl-2 expression was detected by immunohistochemistry method. RESULTS AND CONCLUSION: Based on cel morphology observation and flow cytometry identification, high-purity bone marrow mesenchymal stem cells could be obtained using rat femoral bone marrow adherent culture. Cel growth curve showed that normal rat bone marrow mesenchymal stem cells grew faster than those from diabetic rats. At 1 month after transplantation, histomorphological improvement was seen in the infarcted area after transplantation of normal rat bone marrow mesenchymal stem cells as compared with the other two groups. In addition, the Bcl-2 expression in the infarcted area was higher in the normal rat cel group than the the other two groups. These findings indicate that bone marrow mesenchymal stem cells from normal rats grow faster than those from diabetic rats, and the cells from normal rats have better therapeutic effects on myocardial infarction.

Chondrogenic differentiation of bone marrow mesenchymal stem cells / 中国组织工程研究

BACKGROUND: Bone marrow mesenchymal stem cells are important seeded cells for construction of tissue-engineered trachea, but there is no special surface marker. Therefore, identification of bone marrow mesenchymal stem cells is mostly based on morphology, phenotype antigen and the function of differentiation. OBJECTIVE: To explore the feasibility of the tracheal chondrogenic differentiation of bone marrow mesenchymal stem cells under a special condition through isolation, cultivation and identification of bone marrow mesenchymal stem cells. METHODS: Rabbit bone marrow was acquired in the sterile environment to isolate and culture bone marrow mesenchymal stem cells to passage 2 by bone marrow adherence and screening method. Flow cytometry identified the phenotype CD44, CD45 of bone marrow mesenchymal stem cells at passages 1 and 2. Rabbit tracheal samples were acquired in the sterile environment, the tracheal chondrocytes were isolated and cultured by enzyme digestion, and toluidine blue staining was used to detect aggrecan. Bone marrow mesenchymal stem cells were co-cultured with tracheal chondrocytes by Transwel and transforming growth factor β1. Cel morphology was detected under an inverted microscope. Real-time quantitative PCR and toluidine blue staining detected the extracel ular matrix components, such as type Ⅱ col agen and aggrecan.RESULTS AND CONCLUSION: After isolation and culture, cells were spindle and irregular in morphology, and passaged cells thrived that were gathered into a fish-like colony growth. For passage 1 bone marrow mesenchymal stem cells, the positive rates of phenotype antigen CD44 and CD45 were respectively 96.97% and 13.72%; for passage 2 cells, the positive rates of phenotype antigen CD44 and CD45 were 99.11% and 8.54%, respectively. Tracheal chondrocytes were positive for toluidine blue staining. The morphology of induced bone marrow mesenchymal stem cells changed from long fusiform to triangular or irregular shape, indicating the chondrocytes expressed type Ⅱ col agen and aggrecan, and toluidine blue staining was positive. These results showed bone marrow adherence and screening method could acquire bone marrow mesenchymal stem cells, and the purity of passage 2 cells is higher. Under a special condition, bone marrow mesenchymal stem cells have the potential of chondrogenic differentiation, and can be selected as seed cells for construction of tissue-engineered trachea.

Bone marrow mesenchymal stem cells combined with allogeneic bone for cancellous bone defects / 中国组织工程研究

BACKGROUND: Some studies have shown that bone marrow mesenchymal stem cells and al ograft bone have a certain role for repairing bone defects, but the effectiveness on cancel ous bone defects is seldom reported so far. OBJECTIVE: To observe the effectiveness of bone marrow mesenchymal stem cells combined with al ogeneic bone on cancel ous bone defects. METHODS: The models of cancel ous bone defects (0.6 cm×1.2 cm) were made artificial y in both condylus lateralis femoris of New Zealand white rabbits: one side served as model group implanted with combination of bone marrow mesenchymal stem cells and al ogeneic bone, and the other side was considered as control group implanted with al ogeneic bone. RESULTS AND CONCLUSION: The model group was better than the control group in new bone growth and defect repair at 4, 8, 12 weeks after implantation, which was confirmed by general observation, X-ray examination and hematoxylin-eosin staining. There was a large amount of trabecular bone formation and mature lamel ar bone tissue in bone defects of model group by histological observation at 12 weeks after implantation, and bone defects of the model group were repaired basical y; while there were only abundant woven bones in the control group, and bone defects in the control group were not repaired effectively. Scores on Lane-Sandhu’s X-ray combined with histological observation were higher in the model group than the control group (P < 0.05). Biomechanical test showed that the maximum pressure load of the femoral condyle and load/strain ratio in the model group were significantly higher than those in the control group at 12 weeks after implantation (P < 0.05),while the maximum strain and displacement of the model group was lower than that of the control group (P < 0.05). These findings suggest that the combination of bone marrow mesenchymal stem cells and al ogeneic bone is superior to simple al ogeneic bone implantation in the repair of cancel ous bone defects of the femoral condyle.

Duration and Magnitude of Extracellular Signal-Regulated Protein Kinase Phosphorylation Determine Adipogenesis or Osteogenesis in Human Bone Marrow-Derived Stem Cells

PURPOSE: Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs). MATERIALS AND METHODS: Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates. RESULTS: ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)gamma expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARgamma, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARgamma agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types. CONCLUSION: The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.

Duration and Magnitude of Extracellular Signal-Regulated Protein Kinase Phosphorylation Determine Adipogenesis or Osteogenesis in Human Bone Marrow-Derived Stem Cells

PURPOSE: Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs). MATERIALS AND METHODS: Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates. RESULTS: ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)gamma expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARgamma, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARgamma agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types. CONCLUSION: The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.