Kedong Song

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

Culture of neural stem cells in calcium alginate beads

Neural stem cells (NSCs) with the capacity of extensive self‐renewal and multilineage differentiation have attracted more and more attention in research as NSCs will play an important role in the nerve disease treatment and nerve injury repair. The shortage of NSCs, both their sources and their numbers, however, is the biggest challenge for their clinic application, and hence, in vitro culture and expansion of NSCs is vitally important to realize their potentials. In this work, mouse‐derived NSCs were cultured in three‐dimensional calcium alginate beads (Ca‐Alg‐Bs). Gelling conditions, cell density, and cell harvest were determined by the exploration of formation and dissociation parameters for Ca‐Alg‐Bs. Additionally, the recovered and the subsequent induced cells were identified by immunofluorescence staining of Nestin, β‐tubulin, and GFAP. The results show that the 2‐mm diameter Ca‐Alg‐Bs, prepared with 1.5% sodium alginate solution and 3.5% CaCl2 solution and with gelling for 10 min, is suitable for the NSCs culture. The seeding density of 0.8 × 105 cells·mL−1 for the encapsulation of NSCs resulted in the most expansion, and the NSCs almost doubled during the experiment. The average cell recovery rate is over 88.5%, with the Ca‐Alg‐Bs dissolving in 55 mM sodium citrate solution for 10 min. The recovered cells cultured in the Ca‐Alg‐Bs still expressed Nestin and had the capacity of multilineage differentiation into neurons and glial cells and, thus, remained to be NSCs. These results demonstrate that NSC expansion within Ca‐Alg‐Bs is feasible and provides further possibilities for NSC expansion in bioreactors of the scale of clinical relevance.

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.

Fabrication and detection of tissue‐engineered bones with bio‐derived scaffolds in a rotating bioreactor

In order to explore the methods for commercialized bone tissue engineering, engineered bones should be cultivated in bioreactors to realize three‐dimensional culture under well‐defined culture conditions. In the present paper, osteoblasts isolated from the cranium of 1‐month‐old Zelanian rabbits were inoculated on to the BDBS (bio‐derived bone scaffolds) to investigate the three‐dimensional fabrication of engineered bone in an RWVB (rotating‐wall vessel bioreactor). The osteoblasts, after being transfected with green fluorescent protein, were respectively seeded at 2×106 and 1×106 cells·ml−1 on to the BDBS and then cultured in a T‐flask and an RWVB for 1 week. The morphologies and structure of the fabricated bone were investigated by using an inverted microscope, a scanning electron microscope and a laser confocal microscope using the stains haematoxylin/eosin and Toluidine Blue. After being digested from the scaffolds, the cells were assayed with ALP (alkaline phosphatase) stain, von‐Kossa staining on mineralized nodules, type I collagen and bone morphogenetic protein‐2 expression, and the cell expansion and growth curves using different culture methods were quantitatively determined with MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide). Furthermore, cell cycle and apoptosis were detected by using a flow cytometer, and total DNA was also assayed. For a comparative study, cell‐seeded constructs were also cultured under static conditions. The results show that the cell number cultured in the RWVB was five times that in the T‐flask. Bone tissues cultured in the RWVB with two different densities grew well, and the osteoblasts maintained their normal cycle and DNA content. The result demonstrates that, with the stress stimulation in the fluid in the RWVB, the active expression of ALP can be increased, rapid proliferation and differentiation of osteoblasts are possible and the three‐dimensional fabrication of engineered bone could be realized.

Induced pluripotent stem cells generated from human adipose-derived stem cells using a non-viral polycistronic plasmid in feeder-free conditions.

Induced pluripotent stem cells (iPSCs) can be generated from somatic cells by ectopic expression of defined transcription factors (TFs). However, the optimal cell type and the easy reprogramming approaches that minimize genetic aberrations of parent cells must be considered before generating the iPSCs. This paper reports a method to generate iPSCs from adult human adipose-derived stem cells (hADSCs) without the use of a feeder layer, by ectopic expression of the defined transcription factors OCT4, SOX2, KLF4 and C-MYC using a polycistronic plasmid. The results, based on the expression of pluripotent marker, demonstrated that the iPSCs have the characteristics similar to those of embryonic stem cells (ESCs). The iPSCs differentiated into three embryonic germ layers both in vitro by embryoid body generation and in vivo by teratoma formation after being injected into immunodeficient mice. More importantly, the plasmid DNA does not integrate into the genome of human iPSCs as revealed by Southern blotting experiments. Karyotypic analysis also demonstrated that the reprogramming of hADSCs by the defined factors did not induce chromosomal abnormalities. Therefore, this technology provides a platform for studying the biology of iPSCs without viral vectors, and can hopefully overcome immune rejection and ethical concerns, which are the two important barriers of ESC applications.

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.

Fabrication and evaluation of a sustained-release chitosan-based scaffold embedded with PLGA microspheres

Nutrient depletion within three-dimensional (3D) scaffolds is one of the major hurdles in the use of this technology to grow cells for applications in tissue engineering. In order to help in addressing it, we herein propose to use the controlled release of encapsulated nutrients within polymer microspheres into chitosan-based 3D scaffolds, wherein the microspheres are embedded. This method has allowed maintaining a stable concentration of nutrients within the scaffolds over the long term. The polymer microspheres were prepared using multiple emulsions (w/o/w), in which bovine serum albumin (BSA) and poly (lactic-co-glycolic) acid (PLGA) were regarded as the protein pattern and the exoperidium material, respectively. These were then mixed with a chitosan solution in order to form the scaffolds by cryo-desiccation. The release of BSA, entrapped within the embedded microspheres, was monitored with time using a BCA kit. The morphology and structure of the PLGA microspheres containing BSA before and after embedding within the scaffold were observed under a scanning electron microscope (SEM). These had a round shape with diameters in the range of 27-55 μm, whereas the chitosan-based scaffolds had a uniform porous structure with the microspheres uniformly dispersed within their 3D structure and without any morphological change. In addition, the porosity, water absorption and degradation rate at 37 °C in an aqueous environment of 1% chitosan-based scaffolds were (92.99±2.51) %, (89.66±0.66) % and (73.77±3.21) %, respectively. The studies of BSA release from the embedded microspheres have shown a sustained and cumulative tendency with little initial burst, with (20.24±0.83) % of the initial amount released after 168 h (an average rate of 0.12%/h). The protein concentration within the chitosan-based scaffolds after 168 h was found to be (11.44±1.81)×10(-2) mg/mL. This novel chitosan-based scaffold embedded with PLGA microspheres has proven to be a promising technique for the development of new and improved tissue engineering 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.

Numerical simulation of fluid field and in vitro three-dimensional fabrication of tissue-engineered bones in a rotating bioreactor and in vivo implantation for repairing segmental bone defects

In this paper, two-dimensional flow field simulation was conducted to determine shear stresses and velocity profiles for bone tissue engineering in a rotating wall vessel bioreactor (RWVB). In addition, in vitro three-dimensional fabrication of tissue-engineered bones was carried out in optimized bioreactor conditions, and in vivo implantation using fabricated bones was performed for segmental bone defects of Zelanian rabbits. The distribution of dynamic pressure, total pressure, shear stress, and velocity within the culture chamber was calculated for different scaffold locations. According to the simulation results, the dynamic pressure, velocity, and shear stress around the surface of cell-scaffold construction periodically changed at different locations of the RWVB, which could result in periodical stress stimulation for fabricated tissue constructs. However, overall shear stresses were relatively low, and the fluid velocities were uniform in the bioreactor. Our in vitro experiments showed that the number of cells cultured in the RWVB was five times higher than those cultured in a T-flask. The tissue-engineered bones grew very well in the RWVB. This study demonstrates that stress stimulation in an RWVB can be beneficial for cell/bio-derived bone constructs fabricated in an RWVB, with an application for repairing segmental bone defects.

Biodegradable radiopaque iodinated poly(ester urethane)s containing poly(ε-caprolactone) blocks: Synthesis, characterization, and biocompatibility

Biodegradable radiopaque iodinated poly(ester-urethane) (I-PU), consisting of poly(ε-caprolactone) (PCL) diol and iodinated bisphenol A (IBPA), has been successfully synthesized via a coupling reaction of PCL-diisocyanate and IBPA with varying compositions. The IBPA with four iodine atoms per molecule was applied as a chain extender to endow the I-PUs with intrinsic X-ray visibility. The chemical structure and molecular weights of I-PUs were characterized by Fourier transform infrared spectroscopy (FT-IR), proton-nuclear magnetic resonance, and gel permeation chromatography (GPC). The effects of IBPA on the physical properties of I-PUs were systematically studied by means of differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and wide-angle X-ray diffraction (WAXD). The DSC results showed that the crystallization of PCL segments in I-PUs was restrained with increasing amount of IBPA, which was also confirmed by WAXD. In the X-radiography analysis, all the synthesized I-PUs exhibited high radiopacity compared with an aluminum wedge of equivalent thickness. Enzymatic degradation tests showed that the incorporation of IBPA prolonged the degradation of I-PUs and distinct mass loss and degradation happened in the third month. Basic cytocompatibility conducted using rat adipose-derived cells proved that all the I-PUs and their biodegradation products were nontoxic. The radiopaque I-PUs is expected to possess a significant advantage over the traditional polymer counterparts in some related biomedical fields.