Tianqing Liu

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.

Protocatechuic acid promotes cell proliferation and reduces basal apoptosis in cultured neural stem cells

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia oxyphylla, showed anti-oxidant neuroprotective property in our previous study. However, it is still unknown whether PCA have effects on the cultured neural stem cells (NSCs). In this study, we investigated the roles of PCA in the survival and apoptosis of rat NSCs under normal conditions. NSCs obtained from 13.5-day-old rat embryos were propagated as neurospheres and cultured under normal conditions with or without PCA for 4 and 7 days. The cell viability was determined by the cell counting kit-8 (CCK-8) test, while cell proliferation was assayed by bromodeoxyuridine (BrdU) labeling. PCA increased the cellular viability of NSCs and stimulated cell proliferation in a dose- and time-dependent manner. Apoptotic cells were detected after 4 days by observing the nuclear morphological changes and flow cytometric analysis. Compared with the control on both culture days, treatment with PCA effectively reduced the levels of apoptosis of NSCs. At the same time, the reactive oxygen species (ROS) level in NSCs was depressed. In addition, PCA also significantly decreased the activity of elevated caspase-3, indicating that PCA may inhibit apoptosis of NSCs via suppression of the caspase cascade. These results suggest that PCA may be a potential growth inducer and apoptosis inhibitor for NSCs.

Thermodynamic analysis of the effect of the hierarchical architecture of a superhydrophobic surface on a condensed drop state.

Condensed drops usually display a Wenzel state on a superhydrophobic surface (SHS) only with microrough architecture, while Cassie drops easily appear on a surface with micro-nano hierarchical roughness. The mechanism of this is not very clear. It is important to understand how the hierarchical structure affects the states of condensation drops so that a good SHS can be designed to achieve the highly efficient dropwise condensation. In this study, the interface free energy (IFE) of a local condensate, which comes from the growth and combination of numerous initial condensation nuclei, was calculated during its shape changes from the early flat shape to a Wenzel or Cassie state. The final state of a condensed drop was determined by whether the IFE continuously decreased or a minimum value existed. The calculation results indicate that the condensation drops on the surface only with microroughness display a Wenzel state because the IFE curve of a condensed drop first decreases and then increases, existing at a minimum value corresponding to a Wenzel drop. On a surface with proper hierarchical roughness, however, the interface energy curve of a condensed drop will continuously decline until reaching a Cassie state. Therefore, a condensed drop on a hierarchical roughness surface can spontaneously change into a Cassie state. Besides, the states and apparent contact angles of condensed drops on a SHS with different structural parameters published in the literature were calculated and compared with experimental observations. The results show that the calculated condensed drop states are well-coordinated with experimental clarifications. We can conclude that micro-nano hierarchical roughness is the key structural factor for sustaining condensed drops in a Cassie state on a SHS.

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.

Chitosan/gelatin porous scaffolds containing hyaluronic acid and heparan sulfate for neural tissue engineering

The novel chitosan (Cs)/gelatin (Gel) porous scaffolds containing hyaluronic acid (HA) and heparan sulfate (HS) were fabricated via freeze-drying technique, and their physicochemical characteristics including pore size, porosity, water absorption, and in vitro degradation and biocompatibility were investigated. It was demonstrated that the Cs/Gel/HA/HS composite scaffolds had highly homogeneous and interconnected pores with porosity above 96% and average pore size ranging from 90 to 140 μm and a controllable degradation rate. The scanning electron microscopic images, cell viability assay, and fluorescence microscopy observation revealed that the presence of HA and HS in the scaffolds significantly promoted initial neural stem and progenitor cells (NS/PCs) adhesion and supported long-time growth in three-dimensional environment. Moreover, NS/PCs also maintained mutilineage differentiation potentials with enhanced neuronal differentiation upon induction in the Cs/Gel/HA/HS composite scaffolds in relation to Cs/Gel scaffolds. These results indicated that the Cs/Gel/HA/HS composite scaffolds were suitable for neural cells’ adhesion, survival, and growth and could offer new and important options for neural tissue engineering applications.

OPTIMIZATION OF PRIMARY CULTURE CONDITION FOR MESENCHYMAL STEM CELLS DERIVED FROM UMBILICAL CORD BLOOD WITH FACTORIAL DESIGN

Mesenchymal stem cells (MSCs) can not only support the expansion of hematopoietic stem cells in vitro, but also alleviate complications and accelerate recovery of hematopoiesis during hematopoietic stem cell transplantation. However, it proved challenging to culture MSCs from umbilical cord blood (UCB) with a success rate of 20–30%. Many cell culture parameters contribute to this outcome and hence optimization of culture conditions is critical to increase the probability of success. In this work, fractional factorial design was applied to study the effect of cell inoculated density, combination and dose of cytokines, and presence of serum and stromal cells. The cultured UCB‐MSC‐like cells were characterized by flow cytometry and their multilineage differentiation potentials were tested. The optimal protocol was identified achieving above 90% successful outcome: 2 × 106 cells/mL mononuclear cells inoculated in Iscoves modified Dulbeccos medium supplied with 10% FBS, 15 ng/mL IL‐3, and 5 ng/mL Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Moreover, the UCB‐MSC‐like cells expressed MSC surface markers of CD13, CD29, CD105, CD166, and CD44 positively, and CD34, CD45, and human leukocyte antigens‐DR (HLA‐DR) negatively. Meanwhile, these cells could differentiate into osteoblasts, chondrocytes, and adipocytes similarly to MSCs derived from bone marrow. In conclusion, we have developed an efficient protocol for the primary culture of UCB‐MSCs by adding suitable cytokines into the culture system.

Thermoresponsive copolymer nanofilms for controlling cell adhesion, growth, and detachment.

This study reports the development and use of a novel thermoresponsive polymeric nanofilm for controlling cell adhesion and growth at 37 °C, and then cell detachment for cell recovery by subsequent temperature drop to the ambient temperature, without enzymatic cleavage or mechanical scraping. A copolymer, poly(N-isopropylacrylamide-co-hydroxypropyl methacrylate-co-3-(trimethoxysilyl)propyl methacrylate) (abbreviated PNIPAAm copolymer), was synthesized by free radical polymerization. The thermoresponses of the copolymer in aqueous solution were demonstrated by dynamic light scattering (DLS) through detecting the sensitive changes of copolymer aggregation against temperature. The DLS measurements revealed the lower critical solution temperature (LCST) at approximately 30 °C. The PNIPAAm film stability and robustness was provided through silyl cross-linking within the film and with the hydroxyl groups on the substrate surface. Film thickness, stability, and reversibility with respect to temperature switches were examined by spectroscopic ellipsometry (SE), atomic force microscopy (AFM), and contact angle measurements. The results confirmed the high extent of thermosensitivity and structural restoration based on the alterations of film thickness and surface wettability. The effective control of adhesion, growth, and detachment of HeLa and HEK293 cells demonstrated the physical controllability and cellular compatibility of the copolymer nanofilms. These PNIPAAm copolymer nanofilms could open up a convenient interfacial mediation for cell film production and cell expansion by nonenzymatic and nonmechanical cell recovery.

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.