Yanxia Zhu

Adipose-derived stem cell: a better stem cell than BMSC

To further study the proliferation and multi‐differentiation potentials of adipose‐derived stem cells (ADSCs), the cells were isolated with improved methods and their growth curves were achieved with cck‐8. Surface protein expression was analyzed by flow cytometry to characterize the cell phenotype. The multi‐lineage potential of ADSCs was testified by differentiating cells with adipogenic, chondrogenic, osteogenic, and myogenic inducers. The results showed that about 5 × 105 stem cells could be obtained from 400 to 600 mg adipose tissue. The ADSCs can be continuously cultured in vitro for up to 1 month without passage and they have several logarithmic growth phases during the culture period. Also, the flow cytometry analysis showed that ADSCs expressed high levels of stem cell‐related antigens (CD13, CD29, CD44, CD105, and CD166), while did not express hematopoiesis‐related antigens CD34 and CD45, and human leukocyte antigen HLA‐DR was also negative. Moreover, stem cell‐related transcription factors, Nanog, Oct‐4, Sox‐2, and Rex‐1 were positively expressed in ADSCs. The expression of alkaline phosphatase (ALP) was detected in the early osteogenic induction and the calcified nodules were observed by von Kossa staining. Intracellular lipid droplets could be observed by Oil Red staining. Differentiated cardiomyocytes were observed by connexin43 fluorescent staining. In order to obtain more stem cells, we can subculture ADSCs every 14 days instead of the normal 5 days. ADSCs still keep strong proliferation ability, maintain their phenotypes, and have stronger multi‐differentiation potential after 25 passages. Copyright

ADSCs differentiated into cardiomyocytes in cardiac microenvironment

The microenvironment plays a critical role in directing the progression of stem cells into differentiated cells. So we investigated the role that cardiac microenvironment plays in directing this differentiation process. Adipose tissue-derived stem cells (ADSCs) were cultured with cardiomyocytes directly (“co-culture directly”) or by cell culture insert (“co-culture indirectly”). For co-culture indirectly, differentiated ADSCs were collected and identified. For co-culture directly, ADSCs were labeled with carboxyfluorescein succinimidyl ester (CFSE), Fluorescence-activated cell sorting was used to extract and examine the differentiated ADSCs. The ultrastructure and the expression of cardiac specific proteins and genes were analyzed by SEM, TEM, western blotting, and RT-PCR, respectively. Differentiated ADSCs experienced the co-culture presented cardiac ultrastructure and expressed cardiac specific genes and proteins, and the fractions of ADSCs expressing these markers by co-culture directly were higher than those of co-culture indirectly. These data indicate that in addition to soluble signaling molecules, direct cell-to-cell contact is obligatory in relaying the external cues of the microenvironment controlling the differentiation of ADSCs to cardiomyocytes.

Ex vivo expansion of adipose tissue-derived stem cells in spinner flasks

Recent reports indicate that adipose tissue is a novel source of multipotent stem cells that can be used in cell therapy and tissue engineering. However, using the traditional cultivation of adipose tissue-derived stem cells (ADSCs), it is hard to meet the needs of clinical applications. To obtain a large number of ADSCs while retaining their stemness, we seeded ADSCs in collagen/chitosan scaffolds and compared the proliferation of ADSCs in a 3-D static environment in dishes and a 3-D dynamic environment in spinner flask. The growth dynamic parameters of ADSCs were examined using a CCK-8 kit every other day, and the variations of glucose and lactic acid concentrations were analyzed every day. After 14 days, the cells were observed under a scanning electron microscope. The surface markers (CD29, CD34, CD44, CD45, CD73, CD105, CD166 and HLA-DR), the specific transcription factors (Nanog, Oct-4, Sox-2 and Rex-1) and the multi-differentiation potential (adipogenic, osteogenic and chondrogenic) were also assayed to identify the stemness of expanded cells. The results showed that the cells in scaffolds in spinner flask could be expanded by more than 26 times, and they presented better morphology and vitality and stronger differentiation ability than the cells cultivated in scaffolds statically. All the cells maintained stem cell characteristics after proliferation. Therefore, spinner flask cultivation is an easy-to-use, inexpensive system for expanding ADSCs in 3-D scaffolds.

Enhancement of Adipose-Derived Stem Cell Differentiation in Scaffolds with IGF-I Gene Impregnation Under Dynamic Microenvironment

Biochemical and mechanical signals enabling cardiac regeneration can be elucidated by using in vitro tissue engineering models. We hypothesized that human insulin-like growth factor-I (IGF-I) and 3-dimensional (3D) dynamic microenvironment could enhance the survival and differentiation of adipose tissue-derived stem cells (ADSCs). In this study, ADSCs were cultured on 3D porous scaffolds with or without plasmid DNA PIRES2-IGF-I in cardiac media, in static culture dishes, and in a spinning flask bioreactor, respectively. Cell viability, formation of cardiac-like structure, expression of functional proteins, and gene expressions were tested in the cultured constructs on day 14. The results showed that dynamic microenvironment enhanced the release of plasmid DNA; the ADSCs can be transfected by the released plasmid DNA PIRES2-IGF-I in scaffold. IGF-I showed beneficial effects on cellular viability and increase of total protein and also increased the expressions of cardiac-specific proteins and genes in the grafts. It was also demonstrated that dynamic stirring environment could promote the proliferation of ADSCs. Therefore, IGF-I, expressed by ADSCs transfected by DNA PIRES2-IGF-I incorporated into scaffold, and hydrodynamic microenvironment can independently and interactively increase cellular viability and interactively increase the expression of cardiac-specific proteins and genes in the grafts. The results would be useful for developing tissue-engineered grafts for myocardial repair.

Protocatechuic acid from Alpinia oxyphylla promotes migration of human adipose tissue-derived stromal cells in vitro

Human adipose tissue-derived stromal cells (hADSCs) demonstrate promising potential in various clinical applications, including the transplantation to regenerate injured or degenerative tissues. The migration of engrafted hADSCs to the correct site of injure is essential for the curative effect of stem cell therapy. We found that protocatechuic acid (PCA) from Alpinia (A.) oxyphylla could promote the migration capacity of hADSCs through transwell coated with gelatin in vitro. PCA enhanced the cell migration rate in a dose-dependent and time-dependent manner. Meanwhile, RT-PCR and quantitative RT-PCR analysis revealed the elevation of membrane-type matrix metalloproteinase-1 (MT1-MMP) mRNA expression in 1.5 mM PCA-treated hADSCs. In the supernatants of these cells, the active matrix metalloproteinase-2 (MMP-2) increased compared with control cells with zymography. Moreover, the promotion of cell migration by PCA could be effectively and obviously inhibited by anti-MT1-MMP or anti-MMP-2 antibodies. Furthermore, flow cytometric analysis of the cell surface antigens, osteogenic induction, adipogenic induction and cardiomyocyte-like cell induction demonstrated that hADSCs retained their functional characteristics of multipotential mesenchymal progenitors after PCA treatment. These results suggest that PCA from A. oxyphylla promote the migration of hADSCs in vitro, which is partially due to the increased expression of MT1-MMP and the promotion of MMP-2 zymogen activation.

Adipose-derived stem cell: A better stem cell than BMSC

To further study the proliferation and multi-differentiation potentials of adipose-derived stem cells (ADSCs), the cells were isolated with improved methods and their growth curves were achieved with cck-8. Surface protein expression was analyzed by flow cytometry to characterize the cell phenotype. The multi-lineage potential of ADSCs was testified by differentiating cells with adipogenic, chondrogenic, osteogenic, and myogenic inducers. The results showed that about 5x10

Core level regulatory network of osteoblast as molecular mechanism for osteoporosis and treatment

To develop and evaluate the long-term prophylactic treatment for chronic diseases such as osteoporosis requires a clear view of mechanism at the molecular and systems level. While molecular signaling pathway studies for osteoporosis are extensive, a unifying mechanism is missing. In this work, we provide experimental and systems-biology evidences that a tightly connected top-level regulatory network may exist, which governs the normal and osteoporotic phenotypes of osteoblast. Specifically, we constructed a hub-like interaction network from well-documented cross-talks among estrogens, glucocorticoids, retinoic acids, peroxisome proliferator-activated receptor, vitamin D receptor and calcium-signaling pathways. The network was verified with transmission electron microscopy and gene expression profiling for bone tissues of ovariectomized (OVX) rats before and after strontium gluconate (GluSr) treatment. Based on both the network structure and the experimental data, the dynamical modeling predicts calcium and glucocorticoids signaling pathways as targets for GluSr treatment. Modeling results further reveal that in the context of missing estrogen signaling, the GluSr treated state may be an outcome that is closest to the healthy state.

The generation and functional characterization of induced pluripotent stem cells from human intervertebral disc nucleus pulposus cells

Disc degenerative disease (DDD) is believed to originate in the nucleus pulposus (NP) region therefore, it is important to obtain a greater number of active NP cells for the study and therapy of DDD. Human induced pluripotent stem cells (iPSCs) are a powerful tool for modeling the development of DDD in humans, and have the potential to be applied in regenerative medicine. NP cells were isolated from DDD patients following our improved method, and then the primary NP cells were reprogramed into iPSCs with Sendai virus vectors encoding 4 factors. Successful reprogramming of iPSCs was verified by the expression of surface markers and presence of teratoma. Differentiation of iPSCs into NP-like cells was performed in a culture plate or in hydrogel, whereby skin fibroblast derived-iPSCs were used as a control. Results demonstrated that iPSCs derived from NP cells displayed a normal karyotype, expressed pluripotency markers, and formed teratoma in nude mice. NP induction of iPSCs resulted in the expression of NP cell specific matrix proteins and related genes. Non-induced NP derived-iPSCs also showed some NP-like phenotype. Furthermore, NP-derived iPSCs differentiate much better in hydrogel than that in a culture plate. This is a novel method for the generation of iPSCs from NP cells of DDD patients, and we have successfully differentiated these iPSCs into NP-like cells in hydrogel. This method provides a novel treatment of DDD by using patient-specific NP cells in a relatively simple and straightforward manner.

Adipose tissue-derived stem cell expansion on collagen/chitosan scaffolds in bioreactor

Recent reports indicate that adipose tissue is a novel source of multipotent stem cells, which can be used in cell therapy and tissue engineering. However, the static amplification of adipose tissue-derived stem cells(ADSCs) is a time-consuming procedure and prone to contamination, and its hard to meet the need of clinical applications. To obtain the stem cells in a great number of quantity and with stemness well reserved, we plated ADSCs in collagen/chitosan scaffolds and compared the proliferation of ADSCs in three dimensional (3D) static environment and 3D dynamic environment. hADSCs were isolated and cultivated in T-flask, in collagen/chitosan scaffolds statically or in the scaffolds placed in spinner flasks for 21 days. During the culture period, the growth dynamic parameters and viability of ADSCs were examined by CCK-8 kit every other day; metabolic rates of glucose and lactic acid were analyzed every day. After 21 days, the cells were evaluated with scanning electron microscope (SEM) for histological examination of the aggregates. The surface markers (CD13, CD29, CD34, CD44, CD45, CD105, CD166 and HLA-DR), the specific transcription factors (Oct-4, Sox-2, Rex-1) and the multi-differentiation potential (adipogenic, osteogenic and chondrogenic) were also assayed to identify the stemness of proliferated cells. The results showed that in spinner flask, the cells in scaffolds could be expanded by more than ten times, and they presented better morphology and vitality and stronger differentiation ability than cells cultivated in scaffolds statically. And the expansion of the both cases for cells in scaffolds was better than that in T-flask. All cells maintained stem cell characteristics after proliferation. From the results, we found that dynamic spinner flask cultivation of hADSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for expanding hADSCs in 3D scaffolds.