Wenxue Zhao

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

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.

Collagen membranes loaded with collagen-binding human PDGF-BB accelerate wound healing in a rabbit dermal ischemic ulcer model

Studies have shown that exogenous platelet-derived growth factor-BB (PDGF-BB) could accelerate the ulcer healing, but the lack of efficient growth factor delivery system limits its clinical application. Our previous work has demonstrated that the native human PDGF-BB was added a collagen-binding domain (CBD), TKKTLRT, to develop a collagen-based PDGF targeting delivery system. Here, we showed that this CBD-fused PDGF-BB (CBD-PDGF) could bind to collagen membrane efficiently. We used the rabbit dermal ischemic ulcer model to study the effects of CBD-PDGF loaded on collagen membranes. Results revealed that this system maintained a higher concentration and stronger bioactivity of PDGF-BB on the collagen membranes and promoted the re-epithelialization of dermal ulcer wounds, the collagen deposition, and the formation of capillary lumens within the newly formed tissue area. It demonstrated that collagen membranes loaded with collagen-targeting human PDGF-BB could effectively promote ulcer healing.

Improved neovascularization and wound repair by targeting human basic fibroblast growth factor (bFGF) to fibrin

Targeted therapy is a new generation of therapeutics, where two critical factors are involved. One is the particular molecular target, and the other is the specific target-binding drug. In this work, the fibrin, a main component of plasma clot at wound sites, was used as the target for human bFGF, aiming to improve therapeutic neovascularization and wound repair. To endow bFGF with fibrin-targeting ability, a fibrin-binding peptide Kringle1 (K1), derived from human plasminogen, was fused to human bFGF. The recombinant K1bFGF showed high fibrin and plasma-clot-binding ability. When applied to the wound sites with plasma clots, K1bFGF induced robust neovascularization and improved wound healing. To extend the application of K1bFGF to other cases where no plasma clots exist, we developed a fibrin-scaffold/K1bFGF system. This system could induce localized neovascularization by delivery of K1bFGF in a sustained and site-targeting manner, and provide a microenvironment promoting cell growth and tissue regeneration. In summary, we successfully used the pathologic environment fibrin clot as the target for bFGF, and based on which bFGF was designed into a targeting agent by introduction of a fibrin-binding peptide. This provides a potential approach to improve therapeutic neovascularization and wound repair.

The differentiation of rat adipose-derived stem cells into OEC-like cells on collagen scaffolds by co-culturing with OECs

Olfactory ensheathing cells (OECs) transplantation is a promising or potential therapy for spinal cord injury (SCI). However, their clinical use is limited because of the availability. Adipose-derived stem cells (ADSCs) have been identified as an alternative source of adult stem cells in recent years. ADSCs could be differentiated into various mesenchymal tissues cells such as chondrocytes, adipocytes, osteoblasts, and myocytes and also could be differentiated into neural lineages. In this study, we examined the feasibility of using ADSCs as a source of stem cells for the differentiation of OECs by co-culture approach. When co-cultured with OECs, the ADSCs on three-dimensional collagen scaffolds were differentiated into OEC-like cells, with similar morphology and antigenic phenotypes (p75NTR+/Nestin+/GFAP-) of OECs. Co-cultured ADSCs were positive for several important functional markers of mature OECs such as neurotrophic factor GDNF, BDNF and myelin protein PLP and the conditioned medium of OEC-like cells could significantly promote DRG neuron growth and axon sprouting without NGF supporting in contrast to that of the ADSCs. Our results showed that ADSCs had the potential to differentiate into OEC-like cells on the three-dimensional collagen scaffolds in vitro.

Crosslinked Three-Dimensional Demineralized Bone Matrix for the Adipose-Derived Stromal Cell Proliferation and Differentiation

Stem cell-based therapy has been a promising method for tissue regeneration and wound repair. Adult adipose-derived stromal cells (ADSCs) are often used for adipose and bone tissue reconstruction because of their abundant sources and multipotential differentiation ability. When combined with carriers, ADSCs could be useful for constructing tissue substitutes in vitro or facilitating tissue regeneration in vivo. Demineralized bone matrix (DBM) has been used for tissue reconstruction because collagen presents good cell compatibility. However, DBM degrades rapidly when used for three-dimensional ADSC culture. Here DBM was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysulfosuccinimide to investigate whether crosslinked DBM (CRL-DBM) could be used as ADSC carrier. CRL-DBM showed not only improved mechanical property and enhanced stability, but also sustained ADSC proliferation and effective differentiation into adipocytes and bone lineage cells. The results indicated that CRL-DBM may be a suitable ADSC carrier for adipose and bone tissue regeneration.

Phenotypical analysis of adult rat olfactory ensheathing cells on 3-D collagen scaffolds.

Olfactory ensheathing cell (OEC) transplantation is a promising or potential therapy for spinal cord injury (SCI). However, the effects of injecting OECs directly into SCI site have been limited and unsatisfied due to the complexity of SCI. To improve the outcome, proper biomaterials are thought to be helpful since these materials would allow the cells to grow three-dimensionally and guide cell migration. In this paper, we have studied the behavior of OECs in two-dimensional (2-D) condition as well as on three-dimensional (3-D) collagen scaffolds by analyzing their phenotypes such as cell proliferation, apoptosis, morphology, and gene activities of some neurotrophic factors and myelin proteins. OECs proliferation rate was increased on 3-D collagen scaffolds compared to the 2-D culture condition. OECs on 3-D collagen scaffolds also showed less apoptosis. In addition, OECs on 3-D collagen scaffolds maintained the original spindle-shape morphology and P75NTR gene activity. NGF, BDNF, and PLP were found to be upregulated in OECs cultured on 3-D collagen scaffolds by the semi-quantitative RT-PCR approach. The results suggested that 3-D collagen scaffolds provide suitable environments for the OECs to maintain their morphology as well as several important functional phenotypes and all these could be helpful for the effective treatment of SCI.

Collagen scaffolds loaded with collagen-binding NGF-β accelerate ulcer healing

Studies have shown that exogenous nerve growth factor (NGF) accelerates ulcer healing, but the inefficient growth factor delivery system limits its clinical application. In this report, we found that the native human NGF-beta fused with a collagen-binding domain (CBD) could form a collagen-based NGF targeting delivery system, and the CBD-fused NGF-beta could bind to collagen membranes efficiently. Using the rabbit dermal ischemic ulcer model, we have found that this targeting delivery system maintains a higher concentration and stronger bioactivity of NGF-beta on the collagen membranes by promoting peripheral nerve growth. Furthermore, it enhances the rate of ulcer healing through accelerating the re-epithelialization of dermal ulcer wounds and the formation of capillary lumens within the newly formed tissue area. Thus, collagen membranes loaded with collagen-targeting human NGF-beta accelerate ulcer healing efficiently.

Human Basic Fibroblast Growth Factor Fused with Kringle4 Peptide Binds to a Fibrin Scaffold and Enhances Angiogenesis

Appropriate three-dimensional (3D) scaffolds and signal molecules could accelerate tissue regeneration and wound repair. In this work, we targeted human basic fibroblast growth factor (bFGF), a potent angiogenic factor, to a fibrin scaffold to improve therapeutic angiogenesis. We fused bFGF to the Kringle4 domain (K4), a fibrin-binding peptide from human plasminogen, to endow bFGF with specific fibrin-binding ability. The recombinant K4bFGF bound specifically to the fibrin scaffold so that K4bFGF was delivered in a site-specific manner, and the fibrin scaffold provided 3D support for cell migration and proliferation. Subcutaneous implantation of the fibrin scaffolds bound with K4bFGF but not with bFGF induced neovascularization. Immunohistochemical analysis showed significantly more proliferation cells in the fibrin scaffolds incorporated with K4bFGF than in those with bFGF. Moreover, the regenerative tissues were integrated well with the fibrin scaffolds, suggesting its good biocompatibility. In summary, targeted delivery of K4bFGF could potentially improve therapeutic angiogenesis.

The bone-derived collagen containing mineralized matrix for the loading of collagen-binding bone morphogenetic protein-2

Bone tissue-derived biomaterials have often been applied for bone repair because of their similarity to human bone in structure and composition. When combined with growth factors, they could accelerate bone formation. Here, we explore a collagen containing mineralized bone-derived matrix (CCMBM) from bovine bone tissues, which not only maintains proper mechanical strength but also binds to the collagen-binding recombinant human collagen-binding bone morphogenetic protein-2 (CBD-BMP(2)). By analyzing its morphology and composition, we found that CCMBM was porous and mainly composed of calcium compounds. CCMBM could provide mechanical support for bone injury repair. It also showed good biocompatibility and proper degradation rate that would be helpful for bone regeneration. In addition, the intentionally preserved collagen allowed the specific binding of CBD-BMP(2) to CCMBM, and resulted in significantly increased osteogenesis in vivo. The results indicated that the combination of CCMBM with collagen-binding BMP(2) could be emerged into an effective medical device for bone repair.