In vivo assessment of guided neural stem cell differentiation in growth factor immobilized chitosan-based hydrogel scaffolds

In this study, we demonstrate that a unique growth factor-biomaterial system can offer spatial control of growth factors with sustained signaling to guide the specific lineage commitment of neural stem/progenitor cells (NSPCs) in vivo. First, recombinant fusion proteins incorporating an N-terminal biotin tag and interferon-γ (IFN-γ), platelet derived growth factor-AA (PDGF-AA), or bone morphogenic protein-2 (BMP-2) were immobilized to a methacrylamide chitosan (MAC) based biopolymer via a streptavidin linker to specify NSPC differentiation into neurons, oligodendrocytes, or astrocytes, respectively. MAC was mixed with growth factors (immobilized or adsorbed), acrylated laminin, NSPCs, and crosslinked within chitosan conduits. This system mimics regenerative aspects of the central nervous system ECM, which is largely composed of a crosslinked polysaccharide matrix with cell-adhesive regions, and adds the new functionality of protein sequestration. We demonstrated that these growth factors are maintained at functionally significant levels for 28 d in vitro. In the main study, immobilized treatments were compared to absorbed and control treatments after 28 d in vivo (rat subcutaneous). Masson's Trichrome staining revealed that small collagen capsules formed around the chitosan conduits with an average acceptable thickness of 153.07 ± 6.02 μm for all groups. ED-1 staining showed mild macrophage clustering around the outside of chitosan conduits in all treatments with no macrophage invasion into hydrogel portions. Importantly, NSPC differentiation staining demonstrated that immobilized growth factors induced the majority of cells to differentiate into the desired cell types as compared with adsorbed growth factor treatments and controls by day 28. Interestingly, immobilized IFN-γ resulted in neural rosette-like arrangements and even structures resembling neural tubes, suggesting this treatment can lead to guided dedifferentiation and subsequent neurulation.     

bFGF-containing electrospun gelatin scaffolds with controlled nano-architectural features for directed angiogenesis

Current therapeutic angiogenesis strategies are focused on the development of biologically responsive scaffolds that can deliver multiple angiogenic cytokines and/or cells in ischemic regions. Herein, we report on a novel electrospinning approach to fabricate cytokine-containing nanofibrous scaffolds with tunable architecture to promote angiogenesis. Fiber diameter and uniformity were controlled by varying the concentration of the polymeric (i.e. gelatin) solution, the feed rate, needle to collector distance, and electric field potential between the collector plate and injection needle. Scaffold fiber orientation (random vs. aligned) was achieved by alternating the polarity of two parallel electrodes placed on the collector plate thus dictating fiber deposition patterns. Basic fibroblast growth factor (bFGF) was physically immobilized within the gelatin scaffolds at variable concentrations and human umbilical vein endothelial cells (HUVEC) were seeded on the top of the scaffolds. Cell proliferation and migration was assessed as a function of growth factor loading and scaffold architecture. HUVECs successfully adhered onto gelatin B scaffolds and cell proliferation was directly proportional to the loading concentrations of the growth factor (0-100 bFGF ng/mL). Fiber orientation had a pronounced effect on cell morphology and orientation. Cells were spread along the fibers of the electrospun scaffolds with the aligned orientation and developed a spindle-like morphology parallel to the scaffold's fibers. In contrast, cells seeded onto the scaffolds with random fiber orientation, did not demonstrate any directionality and appeared to have a rounder shape. Capillary formation (i.e. sprouts length and number of sprouts per bead), assessed in a 3-D in vitro angiogenesis assay, was a function of bFGF loading concentration (0 ng, 50 ng and 100 ng per scaffold) for both types of electrospun scaffolds (i.e. with aligned or random fiber orientation).     

Engineering surfaces for site-specific vascular differentiation of mouse embryonic stem cells

Differentiation of stem and progenitor cells routinely relies on the application of soluble growth factors, an approach that enables temporal control of cell fate but enables no spatial control of the differentiation process. Angiogenic progenitor cells derived from mouse embryonic stem cells (ESCs) were differentiated here according to the pattern of immobilized vascular endothelial growth factor-A (VEGF). Mouse ESCs engineered to express green fluorescent protein (eGFP) under control of promoter for the receptor tyrosine kinase Flk1 were used. The Flk1+ angiogenic progenitors were selected from day 3 differentiating embryoid bodies based on their expression of eGFP using fluorescence activated cell sorting. Mouse VEGF₁₆₅ was covalently immobilized onto collagen IV (ColIV) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry. A non-cell adhesive layer of photocrosslinkable chitosan was first created, after which VEGF–ColIV was stamped as 100 μm wide lanes on top of the chitosan layer and the Flk1+ angiogenic progenitors were seeded for site-specific differentiation. Lanes stamped with only ColIV served as controls. The results presented here demonstrate that the cultivation of Flk1+ progenitors on surfaces with immobilized VEGF yielded primarily endothelial cells (53 ± 13% CD31 positive and 17 ± 2% smooth muscle actin positive), whereas surfaces without VEGF favored vascular smooth muscle-like cell differentiation (26 ± 17% CD31 positive and 38 ± 9% smooth muscle actin positive).     

The differentiation of O-2A progenitor cells into oligodendrocytes is associated with a loss of inducibility of Ia antigens

Current data suggest that some astrocytes, one of the 3 main types of macroglia in the central nervous system (CNS), can be induced by interferon-gamma (IFN-gamma) to express major histocompatibility complex class II antigens (immune-associated or Ia) and present antigen to T lymphocytes. In contrast, oligodendrocytes, another type of macroglia, cannot be induced to express Ia. The astrocytes which have been shown to express Ia are from a particular glial lineage and are called type-1 astrocytes. The oligodendrocyte-type-2 astrocyte (O-2A) lineage, which gives rise to oligodendrocytes, also gives rise to a second class of astrocytes called type-2 astrocytes and the ability of type-2 astrocytes or the common O-2A progenitor cell to express Ia is not known. We have now found that both type-2 astrocytes and O-2A progenitor cells can be induced to express Ia by IFN-gamma but Ia expression is not induced in oligodendrocytes in parallel cultures. Thus, it appears that differentiation of O-2A progenitor cells into oligodendrocytes is specifically associated with a loss of inducibility of Ia. This apparent loss of the capacity for Ia expression, and presumably antigen presentation, in oligodendrocytes (the cells which produce myelin in the CNS) is of particular interest in view of the ability of immunization of myelin components to produce autoimmune-mediated paralytic disease.     

Tissue engineering of ureteral grafts by seeding urothelial differentiated hADSCs onto biodegradable ureteral scaffolds

The study is aimed to evaluate the differentiation potential of human adipose-derived stem cells (hADSCs) into urothelial lineage, and to assess possibility of constructing ureteral grafts using the differentiated hADSCs and a novel polylactic acid (PLA)/collagen scaffolds. HADSCs were indirectly cocultured with urothelial cells in a transwell coculture system for urothelial differentiation. After 14 days coculturing, differentiation was evaluated by detecting urothelial lineage markers (cytokeratin-18 and uroplakin 2) in mRNA and protein level. Then the differentiated hADSCs were seeded onto PLA/collagen ureteral scaffolds and cultured in vitro for 1 week. The biocompatibility of the scaffolds was tested by scanning electron microscopy (SEM) and MTT analysis. At last, the cell/scafflod grafts were subcutaneously implanted into 4-week-old female athymic mice for 14 days. The results demonstrated that the hADSCs could be efficiently induced into urothelial lineage by indirect coculture. The differentiated cells seeded onto the PLA/collagen ureteral scaffolds survived up to 7 days and maintained proliferation in vitro, which indicated that the scaffolds displayed good biocompatibility. In vivo study showed that the differentiated cells in the grafts survived, formed multiple layers on the scaffolds and expressed urothelial lineage markers. In conclusion, hADSCs may serve as an alternative cell resource in cell-based tissue engineering for ureteral reconstruction. These cells could be employed to construct a model of ureteral engineering grafts and be effectively applied in vivo, which could be a new strategy on ureteral replacement with applicable potential in clinical research. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:2612-2622, 2012.     

激素预处理树突状细胞对哮喘Th2型反应的影响

目的： 研究激素预处理的树突状细胞（DCs）对哮喘DCs与T细胞共培养上清液中T辅助细胞1(Th1)和Th2型细胞因子的影响及其机制。方法: 采集哮喘组和健康组外周血,分别常规培养和加入地塞米松培养至成熟DCs。将2组常规培养和地塞米松预处理的DC-T细胞共培养72 h。流式细胞仪测定2组常规培养或地塞米松培养成熟DCs的表型。酶联免疫吸附试验（ELISA）检测DC-T细胞共培养上清液中白细胞介素-5(IL-5)和干扰素-γ（IFN-γ）的含量。结果: 哮喘组常规培养的DC-T细胞共培养上清液中IL-5水平高于健康组（P<0.01）,其IFN-γ含量较健康组有减少的趋势,但无显著差异（P>0.05）;地塞米松预处理的DC-T细胞上清液中IL-5水平低于常规培养组（P<0.01）。无论哮喘组或健康组,地塞米松预处理的DC-T细胞共培养上清液中IFN-γ水平均低于未用地塞米松预处理的DC-T细胞共培养上清液（P<0.01）。2组地塞米松预处理的DCs均部分抑制了肿瘤坏死因子-α(TNF-α)所诱导的DCs表型CD83上调（P<0.01）,而上调了CD14的表达（P<0.01）。结论: 哮喘患者常规培养的DCs与同种自体T细胞共培养后呈Th2型反应,地塞米松预处理的DCs可使Th2型反应减弱,其机制可能与地塞米松影响DCs的分化、成熟有关。     

The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages

Due to advances in stem cell biology, embryonic stem (ES) cells can be induced to differentiate into a particular mature cell lineage when cultured as embryoid bodies. Although transplantation of ES cells-derived neural progenitor cells has been demonstrated with some success for either spinal cord injury repair in small animal model, control of ES cell differentiation into complex, viable, higher ordered tissues is still challenging. Mouse ES cells have been induced to become neural progenitors by adding retinoic acid to embryoid body cultures for 4 days. In this study, we examine the use of electrospun biodegradable polymers as scaffolds not only for enhancing the differentiation of mouse ES cells into neural lineages but also for promoting and guiding the neurite outgrowth. A combination of electrospun fiber scaffolds and ES cells-derived neural progenitor cells could lead to the development of a better strategy for nerve injury repair.     

Human adipose tissue-derived stromal cell therapy prevents bone loss in ovariectomized nude mouse

Osteoporosis is a skeletal disorder characterized by reduced bone mineral density (BMD) and increased risk of fracture. We studied the effects of cell therapy of human adipose tissue-derived stromal cell (ADSC) on ovariectomy-induced bone loss in T cell deficient nude mice. Twelve-week-old female nude mice underwent ovariectomy and were treated with ADSC, estrogen, or phosphate buffered saline (PBS). Whole body BMD revealed that treatment of ADSC was more protective against ovariectomy-induced attenuation in bone mass gain compared with PBS control after cell therapy (8.4±1.1 vs. 2.4%±1.4%, p<0.05 at 4 weeks, 13.7±1.3 vs. 7.7%±1.8%, p<0.05 at 8 weeks) and this effect was comparable to that of estrogen. μCT analysis revealed that the effect of ADSCs was specific to trabecular bone. Serum osteocalcin levels were increased 4 weeks after ovariectomy and treatment with ADSCs (76.4±11.6 ng/mL) increased osteocalcin to a greater extent when compared with estrogen (63.1±6.7 ng/mL, p<0.05) or PBS treatment (58.0±9.2 ng/mL, p<0.05). Flow cytometry analysis for PKH26-labeled ADSCs and quantitative real-time PCR analysis for human β-globin from bone revealed that transplanted ADSCs were trafficking in bone 48 h after injection and subsequently disappeared. There was no evidence of long-term engraftment of infused ADSCs in bone. In vitro, treatment with ADSC-conditioned medium enhanced osteogenic differentiation in stromal cells and preosteoblasts. These results suggest that cell therapy of ADSCs protects against ovariectomy-induced bone loss in nude mice in a paracrine manner.     

Edg-8 receptors are preferentially expressed in oligodendrocyte lineage cells of the rat CNS

The messenger RNA for endothelial differentiation gene 8 receptors is known to be expressed almost exclusively in the rat CNS, but the nature of the expressing cells has not been defined. Using an antibody specific for endothelial differentiation gene 8, we investigated the immunohistochemical localization of endothelial differentiation gene 8 receptors in the rat CNS. Immunopositive staining was detected in a subset of glial cells distributed throughout the brain and spinal cord, including both gray and white matter, but not in the dorsal root ganglion. The distribution and morphological similarity in comparative immunostaining for endothelial differentiation gene 8 and various glial markers suggested that endothelial differentiation gene 8 is preferentially expressed in NG2-positive oligodendrocyte progenitor cells in adult rat brains. Counts of endothelial differentiation gene 8-positive cells and NG2-positive cells in the forebrain revealed that a subset of NG2-positive cells was endothelial differentiation gene 8-positive, and that the ratio of endothelial differentiation gene 8-positive cells to NG2-positive cells varied from region to region. In 17-day-old embryonic brains, the endothelial differentiation gene 8 distribution was similar to that of an oligodendrocytic marker, 2',3'-cyclic nucleotide 3'-phosphodiesterase. These data suggest that endothelial differentiation gene 8 receptors are preferentially expressed in oligodendrocyte lineage cells including oligodendrocyte progenitor cells and immature/maturating oligodendrocytes in rat CNS, and that they might have important functions in oligodendrocytic maturation and myelination.     

The effect of substrate stiffness on adult neural stem cell behavior

Adult stem cells reside in unique niches that provide vital cues for their survival, self-renewal and differentiation. In order to better understand the contribution of substrate stiffness to neural stem/progenitor cell (NSPC) differentiation and proliferation, a photopolymerizable methacrylamide chitosan (MAC) biomaterial was developed. Photopolymerizable MAC is particularly compelling for the study of the central nervous system stem cell niche because Young's elastic modulus (E_Y) can be tuned from less than 1 kPa to greater than 30 kPa. Additionally, the numerous free amine functional groups enable inclusion of biochemical signaling molecules that, together with the mechanical environment, influence cell behavior. Herein, NSPCs proliferated on MAC substrates with Young's elastic moduli below 10 kPa and exhibited maximal proliferation on 3.5 kPa surfaces. Neuronal differentiation was favored on the softest surfaces with E_Y < 1 kPa as confirmed by both immunohistochemistry and qRT-PCR. Oligodendrocyte differentiation was favored on stiffer scaffolds (>7 kPa); however, myelin oligodendrocyte glycoprotein (MOG) gene expression suggested that oligodendrocyte maturation and myelination was best on <1 kPa scaffolds where more mature neurons were present. Astrocyte differentiation was only observed on <1 and 3.5 kPa surfaces and represented less than 2% of the total cell population. This work demonstrates the importance of substrate stiffness to the proliferation and differentiation of adult NSPCs and highlights the importance of mechanical properties to the success of scaffolds designed to engineer central nervous system tissue.     

IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia

The differentiation of bone marrow-derived progenitor cells into monocytes, tissue macrophages and some dendritic cell (DC) subtypes requires the growth factor CSF1 and its receptor, CSF1R. Langerhans cells (LCs) and microglia develop from embryonic myeloid precursor cells that populate the epidermis and central nervous system (CNS) before birth. Notably, LCs and microglia are present in CSF1-deficient mice but absent from CSF1R-deficient mice. Here we investigated whether an alternative CSF1R ligand, interleukin 34 (IL-34), is responsible for this discrepancy. Through the use of IL-34-deficient (Il34(LacZ/LacZ)) reporter mice, we found that keratinocytes and neurons were the main sources of IL-34. Il34(LacZ/LacZ) mice selectively lacked LCs and microglia and responded poorly to skin antigens and viral infection of the CNS. Thus, IL-34 specifically directs the differentiation of myeloid cells in the skin epidermis and CNS.     

Tissue engineering of small-diameter vascular grafts by endothelial progenitor cells seeding heparin-coated decellularized scaffolds

Successful construction of a small-diameter bioartificial vascular graft remains a great challenge. This study reports on novel tissue engineering vascular grafts (TEVGs) constructed by endothelial progenitor cells and heparin-coated decellularized vessels (DV). The DVs were fabricated from canine carotid arteries with observable depletion of cellular components. After heparin coating, the scaffolds possessed excellent antithrombogeneity. Canine endothelial progenitor cells harvested from peripheral blood were expanded and seeded onto heparin-coated DVs and cocultured in a custom-made bioreactor to construct TEVGs. Thereafter, the TEVGs were implanted into the carotid arteries of cell-donor dogs. After 3 months of implantation, the luminal surfaces of TEVGs exhibited complete endothelium regeneration, however, only a few disorderly cells and thrombosis overlaid the luminal surfaces of control DVs grafts, and immunofluorescent staining showed that the seeded cells existed in the luminal sides and the medial parts of the explanted TEVGs and partially contributed to the endothelium formation. Specifically, TEVGs exhibited significantly smaller hyperplastic neointima area compared with the DVs, not only at midportion (0.64 ± 0.08 vs. 2.13 ± 0.12 mm(2) , p < 0.001), but also at anastomotic sites (proximal sites, 1.03 ± 0.09 vs. 3.02 ± 0.16 mm(2), p < 0.001; distal sites, 1.84 ± 0.15 vs. 3.35 ± 0.21 mm(2), p < 0.001). Moreover, TEVGs had a significantly higher patency rate than the DVs after 3 months of implantation (19/20 vs. 12/20, p < 0.01). Overall, this study provided a new strategy to develop small-diameter TEVGs with excellent biocompatibility and high patency rate.     

Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2-Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2-Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame-shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.     

Immobilized sonic hedgehog N-terminal signaling domain enhances differentiation of bone marrow-derived mesenchymal stem cells

The signaling domain of Sonic hedgehog (Shh), a potent upstream regulator of cell fate that has been implicated in osteoblast differentiation from undifferentiated mesenchymal cells in its endogenous form, was investigated in an immobilized form as a means for accelerating differentiation of uncommitted cells to the osteoblast phenotype. A recombinant cysteine-modified N-terminal Shh (mShh) was synthesized, purified, and immobilized onto interpenetrating polymer network (IPN) surfaces also grafted with a bone sialoprotein-derived peptide containing the Arg-Gly-Asp (RGD) sequence (bsp-RGD (15)), at calculated densities of 2.42 and 10 pmol/cm2, respectively. The mitogenic effect of mShh was dependent on the mode of presentation, as surfaces with immobilized mShh and bsp-RGD (15) had no effect on the growth rate of rat bone marrow-derived mesenchymal stem cells (BMSCs), while soluble mShh enhanced cell growth compared to similar surface without mShh supplementation. In conjunction with media supplemented with bone morphogenetic protein-2 and -4, mShh and bsp-RGD (15)-grafted IPN surfaces enhanced the alkaline phosphatase activity of BMSCs compared with tissue culture polystyrene and bsp-RGD (15)-grafted IPN surfaces supplemented with soluble mShh, indicating enhanced osteoblast differentiation. The adhesive peptide bsp-RGD (15) was necessary for cell attachment and proliferation, as well as differentiation in response to immobilized mShh. The addition of immobilized Shh substantially improved the differentiation of uncommitted BMSCs to the osteoblast lineage, and therefore warrants further testing in vivo to examine the effect of the stated biomimetic system on peri-implant bone formation and implant fixation.     

The effects of soluble growth factors on embryonic stem cell differentiation inside of fibrin scaffolds

The goal of this research was to determine the effects of different growth factors on the survival and differentiation of murine embryonic stem cell-derived neural progenitor cells (ESNPCs) seeded inside of fibrin scaffolds. Embryoid bodies were cultured for 8 days in suspension, retinoic acid was applied for the final 4 days to induce ESNPC formation, and then the EBs were seeded inside of three-dimensional fibrin scaffolds. Scaffolds were cultured in the presence of media containing different doses of the following growth factors: neurotrophin-3 (NT-3), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-AA, ciliary neurotrophic factor, and sonic hedgehog (Shh). The cell phenotypes were characterized using fluorescence-activated cell sorting and immunohistochemistry after 14 days of culture. Cell viability was also assessed at this time point. Shh (10 ng/ml) and NT-3 (25 ng/ml) produced the largest fractions of neurons and oligodendrocytes, whereas PDGF (2 and 10 ng/ml) and bFGF (10 ng/ml) produced an increase in cell viability after 14 days of culture. Combinations of growth factors were tested based on the results of the individual growth factor studies to determine their effect on cell differentiation. The incorporation of ESNPCs and growth factors into fibrin scaffolds may serve as potential treatment for spinal cord injury.     

The Regulatory Role of IFN-γ on the Proliferation and Differentiation of Hematopoietic Stem and Progenitor Cells

The replenishment of all blood cell lineages is hierarchically organized by the process of hematopoiesis, which is based on the differentiation pathways of hematopoietic stem and progenitor cells (HSPCs). Due to the ability to balance between self-renewal and differentiation, hematopoietic stem cells (HSCs) can generate the appropriate cell type that is required by the immune system and peripheral blood in response to physiological or pathological conditions. Numerous studies have shown that some proinflammatory cytokines contribute to the regulation of the various hematopoietic compartments. Of these, IFN-γ is a type II interferon primarily produced by T cells and natural killer cells, and plays a major role in the defense against invading pathogens and transformed cancer cells; moreover, a growing amount of research indicates that it exerts negative or positive regulatory effect on hematopoiesis. Although IFN-γ is a widely regarded negative regulator of HSC proliferation, it also participates in some chronic infections or hematological malignancies that induce bone marrow failure. Recent studies have demonstrated unexpected effects of IFN-γ, including the promotion of HSC formation and the stimulation of myelopoiesis. Here, we review the direct and indirect effects of IFN-γ on hematopoiesis, as well as the underlying signaling mechanisms of how IFN-γ modulates the self-renewal, cell cycle entry, and proliferation of HSCs. Next, we describe how IFN-γ affects different stages of the lineage differentiation from HSCs. Finally, we discuss the relationship between IFN-γ and compensatory extramedullary hematopoiesis, as well as some related clinical diseases.     

Growth factor priming of synovium-derived stem cells for cartilage tissue engineering

This study investigated the potential use of synovium-derived stem cells (SDSCs) as a cell source for cartilage tissue engineering. Harvested SDSCs from juvenile bovine synovium were expanded in culture in the presence (primed) or absence (unprimed) of growth factors (1?ng/mL transforming growth factor-β(1), 10?ng/mL platelet-derived growth factor-ββ, and 5?ng/mL basic fibroblast growth factor-2) and subsequently seeded into clinically relevant agarose hydrogel scaffolds. Constructs seeded with growth factor-primed SDSCs that received an additional transient application of transforming growth factor-β(3) for the first 21 days (release) exhibited significantly better mechanical and biochemical properties compared to constructs that received sustained growth factor stimulation over the entire culture period (continuous). In particular, the release group exhibited a Young's modulus (267±96?kPa) approaching native immature bovine cartilage levels, with corresponding glycosaminoglycan content (5.19±1.45%ww) similar to native values, within 7 weeks of culture. These findings suggest that SDSCs may serve as a cell source for cartilage tissue engineering applications.