Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro

Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects.     

Paracrine Effect of Mesenchymal Stem Cells Derived from Human Adipose Tissue in Bone Regeneration

Mesenchymal stem cell (MSC) transplantation has proved to be a promising strategy in cell therapy and regenerative medicine. Although their mechanism of action is not completely clear, it has been suggested that their therapeutic activity may be mediated by a paracrine effect. The main goal of this study was to evaluate by radiographic, morphometric and histological analysis the ability of mesenchymal stem cells derived from human adipose tissue (Ad-MSC) and their conditioned medium (CM), to repair surgical bone lesions using an in vivo model (rabbit mandibles). The results demonstrated that both, Ad-MSC and CM, induce bone regeneration in surgically created lesions in rabbit''s jaws, suggesting that Ad-MSC improve the process of bone regeneration mainly by releasing paracrine factors. The evidence of the paracrine effect of MSC on bone regeneration has a major impact on regenerative medicine, and the use of their CM can address some issues and difficulties related to cell transplants. In particular, CM can be easily stored and transported, and is easier to handle by medical personnel during clinical procedures.     

Efficient Generation of Neural Stem Cell-Like Cells from Rat Adipose Derived Stem Cells After Lentiviral Transduction with Green Fluorescent Protein

Neural stem cells (NSCs) can be isolated from nervous tissues or derived from embryonic stem cells. However, their procurement for clinical applications is limited, and there is a need for alternative types of cell that have NSCs properties. In the present study, the differentiation potential of rat adipose-derived stem cells (ADSCs) was evaluated by infecting these cells with a lentiviral vector-encoding green fluorescent protein (GFP). ADSCs transduced with lentivirus were able to generate NSC-like cells, without any effects on their growth, phenotype, and normal differentiation potential. NSC-like cells derived from ADSCs formed neurospheres and expressed high levels of the neural progenitor marker nestin. In the absence of selected growth factors, these neurospheres differentiated into neurons expressing NeuN and MAP2 and GFAP-expressing glia, as determined by immunocytochemistry, Western blotting, and quantitative real-time polymerase chain reaction. These results demonstrate that ADSCs can be induced to generate neurospheres that have NSC-like properties and may thus constitute a potential source of cells in stem cell therapy for neurological disorders.     

胚胎干细胞体外分化为造血干细胞

造血干细胞移植已成为治疗白血病、再生障碍性贫血、重症免疫缺陷征、地中海贫血、急性放射病、某些恶性实体瘤和淋巴瘤等造血及免疫系统功能障碍性疾病的成熟技术和重要手段,另外这一技术还被尝试用于治疗艾滋病,已取得积极的效果。但是由于移植需要配型相同的供体,并且过程复杂,使得造血干细胞移植因缺少配型相同的供体来源以及费用昂贵而不能被广泛应用。胚胎干细胞是一种能够在体外保持未分化状态并且能进行无限增殖的细胞,在适合条件下能够分化为体内各种类型的细胞,研究胚胎干细胞分化为造血干细胞,不仅可作为研究动物的早期造血发生的模型,而且可以增加造血干细胞的来源,还可以通过基因剔除、治疗性克隆等方法来解决移植排斥的问题,从而为造血干细胞移植的发展扫除了障碍,因此有着重要的研究价值和应用前景。现对胚胎干细胞体外分化为造血干细胞的诱导方法,诱导过程中的调控机制,并对胚胎干细胞分化为造血干细胞的存在问题和发展前景进行讨论。     

Induction of Osteogenic Differentiation of Adipose Derived Stem Cells by Microstructured Nitinol Actuator-Mediated Mechanical Stress

The development of large tissue engineered bone remains a challenge in vitro, therefore the use of hybrid-implants might offer a bridge between tissue engineering and dense metal or ceramic implants. Especially the combination of the pseudoelastic implant material Nitinol (NiTi) with adipose derived stem cells (ASCs) opens new opportunities, as ASCs are able to differentiate osteogenically and therefore enhance osseointegration of implants. Due to limited knowledge about the effects of NiTi-structures manufactured by selective laser melting (SLM) on ASCs the study started with an evaluation of cytocompatibility followed by the investigation of the use of SLM-generated 3-dimensional NiTi-structures preseeded with ASCs as osteoimplant model. In this study we could demonstrate for the first time that osteogenic differentiation of ASCs can be induced by implant-mediated mechanical stimulation without support of osteogenic cell culture media. By use of an innovative implant design and synthesis via SLM-technique we achieved high rates of vital cells, proper osteogenic differentiation and mechanically loadable NiTi-scaffolds could be achieved.     

Adhesion,Vitality and Osteogenic Differentiation Capacity of Adipose Derived Stem Cells Seeded on Nitinol Nanoparticle Coatings

Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells.     

Human Adipose Tissue Derived Mesenchymal Stem Cells Aggravate Chronic Cyclosporin Nephrotoxicity by the Induction of Oxidative Stress

The aim of this study was to investigate whether hATMSCs protect against cyclosporine (CsA)-induced renal injury. CsA (7.5 mg/kg) and hATMSCs (3×10⁶/5 mL) were administered alone and together to rats for 4 weeks. The effect of hATMSCs on CsA-induced renal injury was evaluated by assessing renal function, interstitial fibrosis, infiltration of inflammatory cells, and apoptotic cell death. Four weeks of CsA-treatment produced typical chronic CsA-nephropathy. Combined treatment with CsA and hATMSCs did not prevent these effects and showed a trend toward further renal deterioration. To evaluate why hATMSCs aggravated CsA-induced renal injury, we measured oxidative stress, a major mechanism of CsA-induced renal injury. Both urine and serum 8-hydroxydeoxyguanosine(8-OHdG) levels were higher in the CsA+hATMSCs group than in the CsA group (P<0.05). An in vitro study showed similar results. Although the rate of apoptosis did not differ significantly between HK-2 cells cultured in hATMSCs-conditioned medium and those cultured in DMEM, addition of CsA resulted in greater apoptosis in HK-2 cells cultured in hATMSCs-conditioned medium. Addition of CsA increased oxidative stress in the hATMSCs-conditioned medium. The results of our study suggest that treatment with hATMSCs may aggravate CsA-induced renal injury because hATMSCs cause oxidative stress in the presence of CsA.     

Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic, Androgenetic and Fertilized Blastocysts

MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions. In this study, miRNA and messenger RNA (mRNA) profiles were examined by Illumina microarray in mouse embryonic stem cells (ESCs) derived from parthenogenetic, androgenetic, and fertilized blastocysts. The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression. Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs (aESCs) and fertilized ESCs (fESCs), a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs (pESCs) and fESCs, and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs. In addition, a total of 575, 5 and 376 miRNA-mRNA target pairs were observed in aESCs vs. fESCs, pESCs vs. fESCs, and aESCs vs. pESCs, respectively. Furthermore, 15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR. Finally, transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3, Dyrk1b, and Prrg2 mRNA, inhibition of miR-363 increased the expression of Nfat5 and Soat1 mRNA, and inhibition of miR-883b-5p increased Nfat5, Tacstd2, and Ppapdc1 mRNA. These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.     

Human Adipose Tissue-Derived Mesenchymal Stem Cells Target Brain Tumor-Initiating Cells

In neuro-oncology, the biology of neural stem cells (NSCs) has been pursued in two ways: as tumor-initiating cells (TICs) and as a potential cell-based vehicle for gene therapy. NSCs as well as mesenchymal stem cells (MSCs) have been reported to possess tumor tropism capacities. However, there is little data on the migratory capacity of MSCs toward brain tumor-initiating cells (BTICs). This study focuses on the ability of human adipose tissue derived MSCs (hAT-MSCs) to target BTICs and their crosstalk in the microenvironment. BTICs were isolated from three different types of brain tumors. The migration capacities of hAT-MSCs toward BTICs were examined using an in vitro migration assay and in vivo bioluminescence imaging analysis. To investigate the crosstalk between hAT-MSCs and BTICs, we analyzed the mRNA expression patterns of cyto-chemokine receptors by RT-qPCR and the protein level of their ligands in co-cultured medium. The candidate cyto-chemokine receptors were selectively inhibited using siRNAs. Both in vitro and in vivo experiments showed that hAT-MSCs possess migratory abilities to target BTICs isolated from medulloblastoma, atypical teratoid/rhabdoid tumors (AT/RT) and glioblastoma. Different types of cyto-chemokines are involved in the crosstalk between hAT-MSCs and BTICs (medulloblastoma and AT/RT: CXCR4/SDF-1, CCR5/RANTES, IL6R/IL-6 and IL8R/IL8; glioblastoma: CXCR4/SDF-1, IL6R/IL-6, IL8R/IL-8 and IGF1R/IGF-1). Our findings demonstrated the migratory ability of hAT-MSCs for BTICs, implying the potential use of MSCs as a delivery vehicle for gene therapy. This study also confirmed the expression of hAT-MSCs cytokine receptors and the BTIC ligands that play roles in their crosstalk.     

兔骨髓干细胞和脂肪干细胞增殖和分化能力的比较

目的:评价两种不同来源的间充质干细胞,即骨髓干细胞(BMSC)和脂肪干细胞(ADSC)增殖和分化能力,为其将来更好的应用于细胞治疗和组织工程提供一定的理论支持和依据.方法:分别取同一只兔的髂骨骨髓和其腹股沟脂肪作为BMSC和ADSC的来源,分离培养后比较其生物学特性包括细胞的增殖能力(MTT),成骨成脂分化潜能,细胞表面抗原标记物.结果:BMSC类似纤维细胞样,呈长梭形紧密排列;而ADSC细胞整体呈旋涡状排列;但ADSC倍增时间更短,增殖速度更快;两者都具有向成骨成脂分化的潜能,但BMSC的成骨能力更好.结论:ADSC有更短地倍增时间和更快地增殖速度;两者都具有向成骨成脂分化的潜能,但相较于ADSC更强的成脂分化能力,BMSC的成骨分化能力更好.两者都可以作为组织工程的种子细胞.     

Retinal Electrophysiological Effects of Intravitreal Bone Marrow Derived Mesenchymal Stem Cells in Streptozotocin Induced Diabetic Rats

Diabetic retinopathy is the most common cause of legal blindness in developed countries at middle age adults. In this study diabetes was induced by streptozotocin (STZ) in male Wistar albino rats. After 3 months of diabetes, rights eye were injected intravitreally with green fluorescein protein (GFP) labelled bone marrow derived stem cells (BMSC) and left eyes with balanced salt solution (Sham). Animals were grouped as Baseline (n = 51), Diabetic (n = 45), Diabetic+BMSC (n = 45 eyes), Diabetic+Sham (n = 45 eyes), Healthy+BMSC (n = 6 eyes), Healthy+Sham (n = 6 eyes). Immunohistology analysis showed an increased retinal gliosis in the Diabetic group, compared to Baseline group, which was assessed with GFAP and vimentin expression. In the immunofluorescence analysis BMSC were observed to integrate mostly into the inner retina and expressing GFP. Diabetic group had prominently lower oscillatory potential wave amplitudes than the Baseline group. Three weeks after intravitreal injection Diabetic+BMSC group had significantly better amplitudes than the Diabetic+Sham group. Taken together intravitreal BMSC were thought to improve visual function.     

Neurogenic Effects of Cell-Free Extracts of Adipose Stem Cells

Stem-cell-based therapies are regarded as promising treatments for neurological disorders, and adipose-derived stem cells (ASCs) are a feasible source of clinical application of stem cell. Recent studies have shown that stem cells have a therapeutic potential for use in the treatment of various illnesses through paracrine action. To examine the effects of cell components of ASCs on neural stem cells (NSCs), we treated cell-free extracts of ASCs (CFE-ASCs) containing various components with brain-derived NSCs. To elucidate the effects of CFE-ASCs in NSC proliferation, we treated mouse subventricular zone-derived cultured NSCs with various doses of CFE-ASCs. As a result, CFE-ASCs were found to induce the proliferation of NSCs under conditions of growth factor deprivation in a dose-dependent manner (p<0.01). CFE-ASCs increase the expression of neuron and astrocyte differentiation markers including Tuj-1 (p<0.05) and glial fibrillary acidic protein (p<0.01) without altering the cell’s fate in differentiating NSCs. In addition, treatment with CFE-ASCs induces an increase in neurite numbers (p<0.01) and lengths of NSCs (p<0.05). Furthermore, CFE-ASCs rescue the hydrogen peroxide-induced reduction of NSCs’ viability (p<0.05) and neurite branching (p<0.01). Findings from our study indicate that CFE-ASCs support the survival, proliferation and differentiation of NSCs accompanied with neurite outgrowth, suggesting that CFE-ASCs can modulate neurogenesis in the central nervous system.     

Mesenchymal Stem Cells Derived from Adipose Tissue vs Bone Marrow: In Vitro Comparison of Their Tropism towards Gliomas

Introduction

Glioblastoma is the most common primary malignant brain tumor, and is refractory to surgical resection, radiation, and chemotherapy. Human mesenchymal stem cells (hMSC) may be harvested from bone marrow (BMSC) and adipose (AMSC) tissue. These cells are a promising avenue of investigation for the delivery of adjuvant therapies. Despite extensive research into putative mechanisms for the tumor tropism of MSCs, there remains no direct comparison of the efficacy and specificity of AMSC and BMSC tropism towards glioma.

Methods

Under an IRB-approved protocol, intraoperative human Adipose MSCs (hAMSCs) were established and characterized for cell surface markers of mesenchymal stem cell origin in conjunction with the potential for tri-lineage differentiation (adipogenic, chondrogenic, and osteogenic). Validated experimental hAMSCs were compared to commercially derived hBMSCs (Lonza) and hAMSCs (Invitrogen) for growth responsiveness and glioma tropism in response to glioma conditioned media obtained from primary glioma neurosphere cultures.

Results

Commercial and primary culture AMSCs and commercial BMSCs demonstrated no statistically significant difference in their migration towards glioma conditioned media in vitro. There was statistically significant difference in the proliferation rate of both commercial AMSCs and BMSCs as compared to primary culture AMSCs, suggesting primary cultures have a slower growth rate than commercially available cell lines.

Conclusions

Adipose- and bone marrow-derived mesenchymal stem cells have similar in vitro glioma tropism. Given the well-documented ability to harvest larger numbers of AMSCs under local anesthesia, adipose tissue may provide a more efficient source of MSCs for research and clinical applications, while minimizing patient morbidity during cell harvesting.     

Oligoprogenitor Cells Derived from Spermatogonia Stem Cells Improve Remyelination in Demyelination Model

Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells.