Zhifeng Xiao

The enhancement of cancer stem cell properties of MCF-7 cells in 3D collagen scaffolds for modeling of cancer and anti-cancer drugs

Three-dimensional (3D) culture could partially simulate in vivo conditions. In this work, we developed a 3D collagen scaffold to investigate cellular properties of MCF-7 cells. The porous scaffolds not only induced the diversification of cell morphologies but also extended cell proliferation. The expression of pro-angiogenic growth factors and the transcriptions of matrix metalloproteinases (MMPs) were significantly increased in cells cultured in 3D collagen scaffolds. In addition, 3D collagen scaffolds could generate a cell population with the properties of cancer stem cells (CSCs). The upregulation of EMT markers and the downregulation of the epithelial cell marker were observed in cells cultured in collagen scaffolds. The expression of stem cell markers, including OCT4A and SOX2, and breast cancer stem cell signatures, including SOX4, JAG1 and CD49F, was significantly unregulated in 3D collagen scaffolds. The proportion of cells with CSC-like CD44(+)/CD24(-/low) phenotype was notably increased. High-level expression of CSC-associated properties of MCF-7 cells cultured in 3D was further confirmed by high tumorigenicity in vivo. Moreover, xenografts with 3D cells formed larger tumors. The properties of MCF-7 cells in 3D may have partially simulated their in vivo behaviors. Thus, 3D collagen scaffolds might provide a useful platform for anti-cancer therapeutics and CSC research.

Alternative translation of OCT4 by an internal ribosome entry site and its novel function in stress response.

OCT4 is a pivotal transcription factor in maintaining the pluripotency and self‐renewal capacities of embryonic stem (ES) cells. Human OCT4 can generate two isoforms by alternative splicing, termed OCT4A and OCT4B. OCT4A confers the stemness properties of ES cells, whereas the function of OCT4B is unknown. We present here the diverse protein products and a novel function of OCT4 gene. A single OCT4B mRNA can encode three isoforms by alternative translation initiation at AUG and CUG start codons, respectively. A putative internal ribosome entry site (IRES) has been identified in OCT4B mRNA accounting for the translation mechanism. The OCT4B‐190 is upregulated under stress conditions and it may protect cell against apoptosis under stress. This work evokes the significance to distinguish the biological function of the protein products of OCT4. The OCT4 gene, by the regulation of alternative splicing and alternative translation initiation, may carry out more crucial roles in many biological events. STEM CELLS 2009;27:1265–1275

The effect of collagen-binding NGF-β on the promotion of sciatic nerve regeneration in a rat sciatic nerve crush injury model

Nerve growth factor plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical application. It has demonstrated in our previous work that the native human NGF-beta (NAT-NGF) fused with a collagen-binding domain (CBD) could bind to collagen specifically. Since collagen is the major component of nerve extracellular matrix, we speculated that the collagen-binding NGF would target to nerve cells and improve their regeneration. In this report, we found that the fusion protein could specifically bind to endogenous collagen of the rat sciatic nerves and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that collagen-binding NGF could be retained and concentrated at the nerve injured site to promote nerve repair and enhance function recovery following nerve damage. Thus, the collagen-binding NGF could improve the repair of peripheral nerve injury.

Mammalian target of rapamycin (mTOR) is involved in the neuronal differentiation of neural progenitors induced by insulin.

The fate of neural progenitor cells (NPCs) is determined by many extracellular cues. Among them, insulin and insulin-like growth factor (IGF) family are found to promote the neuronal differentiation of NPCs. Akt activation has been indicated to be responsible for the insulin/IGF-I induced neuronal differentiation. However, the mechanism by which insulin/IGF-I-PI3K-Akt pathway induces neurogenesis of NPCs is not clear. In this study, we have demonstrated that mTOR is involved in the insulin-induced neuronal differentiation. Insulin induces neurogenesis of NPCs in a dose-dependent manner. Phosphorylated mTOR has been up-regulated in a PI3K-Akt dependent manner during NPC differentiation induced by insulin. The specific inhibitor of mTOR, rapamycin, can abrogate the increase of differentiated neurons stimulated by insulin. In addition, this is not the result from the apoptosis of neurons or NPCs. This research has extended the understanding of functions of mTOR and the mechanism of NPC differentiation regulated by insulin.

The use of laminin modified linear ordered collagen scaffolds loaded with laminin-binding ciliary neurotrophic factor for sciatic nerve regeneration in rats

Nerve conduit provides a promising strategy for nerve injury repair in the peripheral nervous system (PNS). However, simply bridging the transected nerve with an empty conduit is hard to satisfy functional recovery. The regenerated axons may disperse during regeneration in the empty lumen, limiting the functional recovery. Our previous work had reported that linear ordered collagen scaffold (LOCS) could be used as a nerve guidance material. Here we cross-linked LOCS fibers with laminin which was a major component of the extracellular matrix in nervous system. Ciliary neurotrophic factor (CNTF) plays a critical role in peripheral nerve regeneration. But the lack of efficient CNTF delivery approach limits its clinical applications. To retain CNTF on the scaffold, a laminin binding domain (LBD) was fused to the N-terminal of CNTF. Compared with NAT-CNTF, LBD-CNTF exhibited specific laminin-binding ability and comparable neurotrophic bioactivity. We combined LBD-CNTF with the laminin modified LOCS fibers to construct a double-functional bio-scaffold. The functional scaffold was filled in silicon conduit and tested in the rat sciatic nerve transection model. Results showed that this functional biomaterial could guide the axon growth, retain more CNTF on the scaffolds and enhance the nerve regeneration as well as functional recovery.

Transplantation of human mesenchymal stem cells loaded on collagen scaffolds for the treatment of traumatic brain injury in rats

Studies have suggested that mesenchymal stem cells (MSCs) have therapeutic effects following traumatic brain injury (TBI). However, cell distribution and survival rate are two major barriers to their success as therapeutic treatment. The improvement of cell therapy using collagen delivery matrices had been reported. However, we know very little about the mechanisms. We labeled human bone marrow-derived mesenchymal stem cells (hMSCs) with a positron emission tomography (PET) tracer, 18F-fluoro-2-deoxy-D-glucose (FDG). hMSCs were transplanted with or without collagen scaffolds into rats with experimental TBI and the whole-body nuclear images were compared. Collagen scaffolds increased the retention of hBMSC in the lesion site and limited its distribution at the transplanted region. Significantly more hMSCs were detected in the brain when transplanted with collagen scaffolds. The results showed collagen scaffolds also efficiently improved cell survival and neurite outgrowth in vivo, resulting in better neural functional recovery. In addition, brain metabolism also improved in the collagen scaffold implanted group, as evaluated by PET. We speculated that collagen scaffolds would improve early engraftment and support the survival of grafted cells post-transplantation.

Nogo-66 Promotes the Differentiation of Neural Progenitors into Astroglial Lineage Cells through mTOR-STAT3 Pathway

Background Neural stem/progenitor cells (NPCs) can differentiate into neurons, astrocytes and oligodendrocytes. NPCs are considered valuable for the cell therapy of injuries in the central nervous system (CNS). However, when NPCs are transplanted into the adult mammalian spinal cord, they mostly differentiate into glial lineage. The same results have been observed for endogenous NPCs during spinal cord injury. However, little is known about the mechanism of such fate decision of NPCs. Methodology/Principal Findings In the present study, we have found that myelin protein and Nogo-66 promoted the differentiation of NPCs into glial lineage. NgR and mTOR-Stat3 pathway were involved in this process. Releasing NgR from cell membranes or blocking mTOR-STAT3 could rescue the enhanced glial differentiation by Nogo-66. Conclusions/Significance These results revealed a novel function of Nogo-66 in the fate decision of NPCs. This discovery could have profound impact on the understanding of CNS development and could improve the therapy of CNS injuries.

Stem-cell-capturing collagen scaffold promotes cardiac tissue regeneration

Stem cell based therapy is coming of age. Besides stem cell transplantation, it has been a goal to use native autologous stem cells for tissue regeneration. However, the recruitment of native autologous stem cells at the targeting site has not been sufficient which limits the clinical application of autologous stem cells. Biomaterials have been increasingly used in tissue repair. They not only serve as scaffolds for cell proliferation, differentiation, and also provide guidance for 3-D reestablishment. In this study, we have attempted to enrich autologous stem cells at the wound site through a stem-cell-capturing collagen scaffold by conjugating with a stem cell specific antibody. Sca-1 is a common surface marker of hematopoietic, cardiac and skeletal muscle stem cells. Due to the interaction of antibody and antigen, Sca-1 positive cells could be enriched to the functional collagen scaffold both in vitro and in vivo. When the functional collagen scaffold is transplanted into C57/BL6 mouse as a patch to repair a surgical heart defect, the regeneration of cardiomyocytes has been observed. Thus, the collagen scaffolds covalently conjugated with stem cell specific antibody could be an effective approach to promote tissue regeneration.

The promotion of neurological recovery in the rat spinal cord crushed injury model by collagen-binding BDNF.

Spinal cord crushed injury is clinically common. Promoting targeted neural regeneration at the crushed site of spinal cord could be important for the repair. It has been demonstrated in our previous work that native human BDNF fused with a collagen-binding domain (CBD-BDNF) can bind to collagen specifically to exert the neurotrophic effect on promoting axonal regeneration. After injury, collagen is highly accumulated at the injury site. We thus speculate that CBD-BDNF will bind to the extracellular matrix collagen and concentrate at the injury site to improve the therapy. Using the rat spinal cord crushed injury model, we have found that CBD-BDNF by one-time intrathecally injection could be retained and concentrated at the injury site for a longer time than native BDNF without collagen-binding domain. CBD-BDNF could promote better neural regeneration and locomotion recovery.

Regeneration of uterine horns in rats by collagen scaffolds loaded with collagen-binding human basic fibroblast growth factor.

Severe damages of uterine endometrium which prevent embryos from implantation and placentation finally often result in infertility or pregnant complications. There is lack of effective treatments due to the limitation of native materials available and complexity of the function and internal environment of uterus. In the present study, a collagen targeting basic fibroblast growth factor (bFGF) delivery system was constructed by a collagen membrane loaded with bFGF fused a collagen-binding domain (CBD) to the N-terminal which limits the diffusion of bFGF from collagen. We tested the bFGF delivery system in rats under the severe uterine damage model (partial rat uterine horn excision/reconstruction), and found this delivery system improved regeneration abilities of uterine endometrium and muscular cells, improved vascularization, as well as better pregnancy outcomes in rats. Therefore, this targeting delivery system may be an effective strategy for uterine tissue regeneration.