Advances in the differentiation of pluripotent stem cells into vascular cells

Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood vessels are related to many disorders like stroke,myocardial infarction,aneurysm,and diabetes,which are important causes of death worldwide.Translational research for new appro-aches to disease modeling and effective treatment is needed due to the huge socio-economic burden on healthcare systems.Although mice or rats have been widely used,applying data from animal studies to human-specific vascular physiology and pathology is difficult.The rise of induced pluripotent stem cells(iPSCs)provides a reliable in vitro resource for disease modeling,regenerative medicine,and drug discovery because they carry all human genetic information and have the ability to directionally differentiate into any type of human cells.This review summarizes the latest progress from the establishment of iPSCs,the strategies for differentiating iPSCs into vascular cells,and the in vivo trans-plantation of these vascular derivatives.It also introduces the application of these technologies in disease modeling,drug screening,and regenerative medicine.Additionally,the application of high-tech tools,such as omics analysis and high-throughput sequencing,in this field is reviewed.

MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4

Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.

Tissue Extracts From Infarcted Myocardium of Rats in Promoting the Differentiation of Bone Marrow Stromal Cells Into Cardiomyocyte-like Cells

Objective To investigate whether cardiac tissue extracts from rats could mimic the cardiac microenvironment and act as a natural inducer in promoting the differentiation of bone marrow stromal cells （BMSCs） into cardiomyocytes. Methods Three kinds of tissue extract or cell lysate [infarcted myocardial tissue extract （IMTE）, normal myocardial tissue extract （NMTE） and cultured neonatal myocardial lysate （NML）] were employed to induce BMSCs into cardiomyocyte-like cells. The cells were harvested at each time point for reverse transcription-polymerase chain reaction （RT-PCR） detection, immunocytochemical analysis, and transmission electron microscopy. Results After a 7-day induction, BMSCs were enlarged and polygonal in morphology. Myofilaments, striated sarcomeres, Z-lines, and more mitochondia were observed under transmission electron microscope. Elevated expression levels of cardiac-specific genes and proteins were also confirmed by RT-PCR and immunocytochemistry. Moreover, IMTE showed a greater capacity of differentiating BMSCs into cardiomyocyte-like cells. Conclusions Cardiac tissue extracts, especially IMTE, can effectively differentiate BMSCs into cardiomyocyte-like cells.

Neuronal differentiation effects of vascular endothelial factor on bone marrow stromal cells

BACKGROUND：Studies have demonstrated that bone marrow stromal cells （BMSCs） undergo neuronal differentiation under certain in vitro conditions.However,very few inducers of BMSC differentiation have been used in clinical application.The effects of vascular endothelial growth factor （VEGF） on in vitro neuronal differentiation of BMSCs remain poorly understood.OBJECTIVE：To investigate the effect of VEGF on neuronal differentiation of BMSCs in vitro,and to determine the best VEGF concentration for experimental induction.DESIGN,TIME AND SETTING：In vitro comparative study was performed at the Central Laboratory and Laboratory of Male Reproductive Medicine,Shenzhen Hospital of Peking University from October 2008 to August 2009.MATERIALS：Recombinant human VEGF165 was purchased from Peprotech Asia,Rehovot,Israel.Neuron-specific enolase （NSE） was purchased from Beijing Biosynthesis Biotechnology,China.METHODS：BMSCs were harvested from adult Sprague Dawley rats.The passaged cells were pre-induced with 10 ng/mL basic fibroblast growth factor for 24 hours,followed by differentiation induction with 0,5,10,and 20 ng/mL VEGF,respectively.MAIN OUTCOME MEASURES：Morphological changes in BMSCs prior to and following VEGF induction.Expression of NSE following induction was determined by immunocytochemistry.RESULTS：Shrunken,round cells,with a strong refraction and thin bipolar or multipolar primary and secondary branches were observed 3 days after induction with 5,10,and 20 ng/mL VEGF.However,these changes were not observed in the control group.At 10 days after induction,the number of NSE-positive cells was greatest in the 10 ng/mL VEGF-treated group （P〈 0.05）.The number of NSE-positive cells was least in the control group at 3 and 10 days post-induction （P〈 0.05）.Moreover,the number of NSE-positive cells was greater at 10 days compared with at 3 days after induction （P〈 0.05）.CONCLUSION：Of the VEGF concentrations tested,10 ng/mL induced the greatest number of neuronal-like cells in vitro from BMSCs.

Prevascularized Micro‑/Nano‑Sized Spheroid/Bead Aggregates for Vascular Tissue Engineering

Efficient strategies to promote microvascularization in vascular tissue engineering,a central priority in regenerative medicine,are still scarce;nano-and micro-sized aggregates and spheres or beads harboring primitive microvascular beds are promising methods in vascular tissue engineering.Capillaries are the smallest type and in numerous blood vessels,which are distributed densely in cardiovascular system.To mimic this microvascular network,specific cell components and proangiogenic factors are required.Herein,advanced biofabrication methods in microvascular engineering,including extrusion-based and droplet-based bioprinting,Kenzan,and biogripper approaches,are deliberated with emphasis on the newest works in prevascular nano-and micro-sized aggregates and microspheres/microbeads.

Wnt7b can replace Ihh to induce hypertrophic cartilage vascularization but not osteoblast differentiation during endochondral bone development

Indian hedgehog （Ihh） is an essential signal that regulates endochondral bone development. We have previously shown that Wnt7b promotes osteoblast differentiation during mouse embryogenesis, and that its expression in the perichondrium is dependent on Ihh signaling. To test the hypothesis that Wnt7b may mediate some aspects of Ihh function during endochondral bone development, we activated Wnt7b expression from the R26-Wnt7b allele with Col2-Cre in the Ihh-/- mouse. Artificial expression of Wnt7b rescued vascularization of the hypertrophic cartilage in the Ihh-/- mouse, but failed to restore orthotopic osteoblast differentiation in the perichondrium. Similarly, Wnt7b did not recover Ihh-dependent perichondral bone formation in the Ihh-/-; Gli3-/- embryo. Interestingly, Wnt7b induced bone formation at the diaphyseal region of long bones in the absence of Ihh, possibly due to increased vascularization in the area. Thus, Ihh-dependent expression of Wnt7b in the perichondrium may contribute to vascularization of the hypertrophic cartilage during endochondral bone development.

Adipokines regulate mesenchymal stem cell osteogenic differentiation

Mesenchymal stem cells(MSCs)can differentiate into various tissue cell types including bone,adipose,cartilage,and muscle.Among those,osteogenic differentiation of MSCs has been widely explored in many bone tissue engineering studies.Moreover,the conditions and methods of inducing osteogenic differentiation of MSCs are continuously advancing.Recently,with the gra-dual recognition of adipokines,the research on their involvement in different pathophysiological processes of the body is also deepening including lipid metabolism,inflammation,immune regulation,energy disorders,and bone homeostasis.At the same time,the role of adipokines in the osteogenic differentiation of MSCs has been gradually described more completely.Therefore,this paper reviewed the evidence of the role of adipokines in the osteogenic differentiation of MSCs,emphasizing bone formation and bone regeneration.

Vascular smooth muscle cell differentiation－2010

Vascular smooth muscle cells have attracted considerable interest as a model for a flexible program of gene expression.This cell type arises throughout the embryo body plan via poorly understood signaling cascades that direct the expression of transcription factors and microRNAs which,in turn,orchestrate the activation of contractile genes collectively defining this cell lineage.The discovery of myocardin and its close association with serum response factor has represented a major break-through for the molecular understanding of vascular smooth muscle cell differentiation.Retinoids have been shown to improve the outcome of vessel wall remodeling following injury and have provided further insights into the molecular circuitry that defines the vascular smooth muscle cell phenotype.This review summarizes the progress to date in each of these areas of vascular smooth muscle cell biology.

Protective effect of tannins from Ficus racemosa in hypercholesterolemia and diabetes induced vascular tissue damage in rats

Objective:To evaluate the protective effect of tannins from Ficus racemosa(F.racemosa) on the lipid profile and antioxidant parameters in high fat meal and streptozotocin induced hypercholestremia associated diabetes model in rats.Methods:The crude tannin fraction was separated from the acetone(70%v/v) bark extract of F.racemosa.Oral administratipn of tannin fraction(TF)(100 & 200 mg/kg body weight) to rats fed with high fat meal for 30 days (4%cholesterol,1%cholic acid,0.5%egg albumin) and injected with streptozotocin(35 mg/ kg i.p.in citrate buffer on 14th day).Results:The administration of TF significantly reverse the increased blood glucose,total cholesterol,triglycerides,low density lipoprotein and also significandy restored the insulin and high density lipoprotein in the serum.In addition tannins significantly restored the activity of antioxidant enzymes such as superoxide dismutase,catalase and decreased the,glutathione peroxidase,and glutathione,thereby restoring the antioxidant status of the organs to almost normal levels.Conclutions:The results of this study show that two different doses of tannin supplementation had a favorable effect on plasma glucose and lipid profile concentrations.It also had an influence on attenuating oxidative stress in diabetic tats.

High-Fat Diets-Induced Metabolic Alterations Alter the Differentiation Potential of Adipose Tissue-Derived Stem Cells

Background: Adipose tissue-derived stem cells (ASC) possess the ability to differentiate into adipocytes or endothelial cells to help in the adipogenesis, vasculogenesis and vascular repair. This study aims at determining the impact of high-fat diets (HFD)-induced type 2 diabetes (T2D) on the differentiation potential of ASC. Results: C57BL/6J male mice were fed a vegetal (VD) or an animal (AD) HFD. Isolation of ACS from mice showing different levels of metabolic alterations reveals that advanced T2D did not affect the number of cells per gram of tissue. Rather, a higher proportion of inflammatory CD36+ cells was identified in HFD fed mice. Despite a marked decreased expression of adipogenic genes (aP2, C/EBPα and PPARγ2), ASC from HFD groups had a higher adipogenic potential and a lower endothelial differentiation potential in vitro compared to control. ASC from the VD group had enhanced cyclin B1 expression and had more adipogenic potential compared to AD group. Conclusion: Our results demonstrate that the metabolic modifications, linked to the nature of fatty acids in diets, modulate the differentiation potential of ASC with increased adipogenesis to the detriment of the endothelial pathway. Results highlight the importance of evaluating the ASC differentiation behavior in a context of autologous cell-based therapy for the repair of vascular tissues in diabetic patients.

Tissue induction, the relationship between biomaterial’s microenvironment and mesenchymal stem cell differentiation

Mesenchymal stem cells (MSCs) have been recognized as the best candidate for tissue engineering, while the mechanism of biomaterial-induced MSCs differentiation is not well understood. Most of research has been focused on chemical signaling of biomaterial in the past, but a variety of non-chemical signals were also proved to play essential roles in cellular behaviors. In this paper, we reviewed the current reports about the effects of different kinds of biomaterial signals on MSCs differentiation.

Effect of screw position on bone tissue differentiation within a fixed femoral fracture

Plate and screw constructs are routinely used in the treatment of long bone fractures. Despite considerable advancements in technology and techniques, there can still be complications in the healing of long bone fractures. Non-unions, delayed unions, and hardware failures are common complications observed in clinical practice following open reduction and internal fixation of fractures [1]. Potential causes of these adverse clinical effects may be disruptive to the periosteal and endosteal blood supply, stress shielding effects, and inadequate mechanical stability. The goal of the present study was to explore the effect of screw position on the fracture healing and formation of new bone tissue with mechanoregulatory algorithms in a computational model. An idealized poroelastic 3D finite element (FE) model of a femur with a 5 mm fracture gap, including a plate-screw construct was developed. Nineteen different plate-screw combinations, created by varying the number and position of screws within the plate, were created to identify a construct with the most favourable attributes for fracture healing. The first phase of the study evaluated constructs through mechanical stress analyses to identify those constructs with high loadsupport capability. The second phase of the study evaluated healing and bone formation with a biphasic mechanoregulatory algorithm to simulate tissue differentiation for fixation within selected constructs. The results of our analysis demonstrated a 4-screw symmetrical construct with the largest distance between screws to provide the most favourable balance of stability and optimized conditions to promote fracture healing.

Fabrication of a Bi-layer Tubular Scaffold Consisted of a Dense Nanofibrous Inner Layer and a Porous Nanoyarn Outer Layer for Vascular Tissue Engineering

Recent years, it has attracted more attentions to increase the porosity and pore size of nanofibrous scaffolds to provide the for the cells to grow into the small-diameter vascular grafts. In this study, a novel bi-layer tubular scaffold with an inner layer and an outer layer was fabricated. The inner layer was random collagen/poly （ L-lactide-co-caprolactone ） I P （ LLA- CL） ] nanofibrous mat fabricated by conventional electrospinning and the outer layer was aligned collagen/P （LLA-CL） nanoyarns prepared by a dynamic liquid dectrospinning method. Fourier transform infrared spectroscopy （FTIR） was used to characterize the chemical structure. Scanning electron microscopy （ SEM ） was employed to observe the morphology of the layers and the cross- sectioned bi-layer tubular scaffold. A liquid displacement method was employed to measure the porosities of the inner and outer layers. Stress-strain curves were obtained to evaluate the mechanical properties of the two different layers and the bi-layer membrane. The diameters of the nanofibers and the nanoyarns were （480 ± 197 ） nm and （ 19.66 ± 4.05 ） μm, respectively. The outer layer had a significantly higher porosity and a larger pore size than those of the inner layer. Furthermore, the bi-layer membrane showed a good mechanical property which was suitable as small-diameter vascular graft. The results indicated that the bi-layer tubular scaffold had a great potential application in small vascular tissue engineering.

Gibberellin promotes cambium reestablishment during secondary vascular tissue regeneration after girdling in an auxin-dependent manner in Populus

Secondary vascular tissue(SVT)development and regeneration are regulated by phytohormones.In this study,we used an in vitro SVT regeneration system to demonstrate that gibberellin(GA)treatment significantly promotes auxin-induced cambium reestablishment.Altering GA content by overexpressing or knocking down ent-kaurene synthase(KS)affected secondary growth and SVT regeneration in poplar.The poplar DELLA gene GIBBERELLIC ACID INSENSITIVE(PtoGAI)is expressed in a specific pattern during secondary growth and cambium regeneration after girdling.Overexpression of PtoGAI disrupted poplar growth and inhibited cambium regeneration,and the inhibition of cambium regeneration could be partially restored by GA application.Further analysis of the PtaDR5:GUS transgenic plants,the localization of PIN-FORMED 1(PIN1)and the expression of auxin-related genes found that an additional GA treatment could enhance the auxin response as well as the expression of PIN1,which mediates auxin transport during SVT regeneration.Taken together,these findings suggest that GA promotes cambium regeneration by stimulating auxin signal transduction.

Preliminary Study on Callus Differentiation of Hermaphrodite Papaya

With calluses of hermaphrodite papaya as the tested material and MS as the basic medium, the effects of different plant growth regulators and their combinations on adventitious bud induction of calluses and rooting induction of sterile buds were investigated. The results showed that 6-BA and TDZ all showed certain induction effect on callus differentiation of papaya; the induction effect of 6-BA was better than that of TDZ, and the optimum concentration of 6-BA was 0.05 mg/L. GA3 could promote the induction effect of 6-BA for on callus differentiation. The optimum medium combination for inducing the callus differentiation of papaya was MS ＋ 6- BA 0.5 mg/L ＋ GA3 1.0 mg/L. Compared to NAA, IBA was more suitable for inducing the rooting of adventitious buds. The optimum rooting-induction medium combination was MS ＋ IBA 0.3 mg/L.

Human hair follicle-derived mesenchymal stem cells:Isolation,expansion,and differentiation

Hair follicles are easily accessible skin appendages that protect against cold and potential injuries.Hair follicles contain various pools of stem cells,such as epithelial,melanocyte,and mesenchymal stem cells(MSCs)that continuously self-renew,differentiate,regulate hair growth,and maintain skin homeostasis.Recently,MSCs derived from the dermal papilla or dermal sheath of the human hair follicle have received attention because of their accessibility and broad differentiation potential.In this review,we describe the applications of human hair follicle-derived MSCs(hHF-MSCs)in tissue engineering and regenerative medicine.We have described protocols for isolating hHF-MSCs from human hair follicles and their culture condition in detail.We also summarize strategies for maintaining hHF-MSCs in a highly proliferative but undifferentiated state after repeated in vitro passages,including supplementation of growth factors,3D suspension culture technology,and 3D aggregates of MSCs.In addition,we report the potential of hHF-MSCs in obtaining induced smooth muscle cells and tissue-engineered blood vessels,regenerated hair follicles,induced red blood cells,and induced pluripotent stem cells.In summary,the abundance,convenient accessibility,and broad differentiation potential make hHF-MSCs an ideal seed cell source of regenerative medical and cell therapy.

Interleukin-24 is correlated with differentiation and lymph node numbers in rectal cancer

AIM:To assess the significance of interleukin(IL)-24 and vascular endothelial growth factor(VEGF)expression in lymph-node-positive rectal cancer. METHODS:Between 1998 and 2005,90 rectal adenocarcinoma patients with lymph node involvement were enrolled.All patients received radical surgery and postoperative pelvic chemoradiotherapy of 50.4-54.0 Gy.Chemotherapy of 5-fluorouracil and leucovorin or levamisole was given intravenously during the first and last week of radiotherapy,and then monthly for about 6 mo.Expression of IL-24 and VEGF was evaluated by immunohistochemical staining of surgical specimens, and their relations with patient characteristics and survival were analyzed.The median follow-up of surviving patients was 73 mo(range:52-122 mo). RESULTS:IL-24 expression was found in 81 out of 90 patients;31 showed weak intensity and 50 showedstrong intensity.VEGF expression was found in 64 out of 90 patients.Negative and weak intensities of IL-24 expression were classified as negative expression for analysis.IL-24 expression was significantly reduced in poorly differentiated tumors in comparison with well or moderately differentiated tumors(P=0.004),N2b to earlier N stages(P=0.016),and stageⅢc to stageⅢ a orⅢb(P=0.028).The number of involved lymph nodes was also significantly reduced in IL-24-positive patients in comparison with IL-24-negative ones. There was no correlation between VEGF expression and patient characteristics.Expression of IL-24 and VEGF was not correlated with survival,but N stage and stages were significantly correlated with survival. CONCLUSION:IL-24 expression was significantly correlated with histological differentiation,and inversely correlated with the degree of lymph node involvement in stageⅢrectal cancer.

TGF-beta 1 Gene-Activated Matrices Regulated the Osteogenic Differentiation of BMSCs

Poly （lactic acid/glycolic acid/asparagic acid-co-polyethylene glycol）（PLGA-[ASP-PEG]） scaffold materials were linked with a novel nonviral vector （K）16GRGDSPC through cross linker Sulfo- LC-SPDP to construct a new type of nonviral gene transfer system. Eukaryotic expressing vector containing transforming growth factor beta 1 （pcDNA3-TGFβ1） was encapsulated by the system. Bone marrow stromal cells （BMSCs） obtained from rabbit were cultured on PLGA-[ASP-PEG] modified by （K）16GRGDSPC and TGF-β1 gene and PLGA-[ASP-PEG] modified by （K）16GRGDSPC and empty vector pcDNA3 as control. The expressions of osteogenic makers of the BMSCs cultured on the TGF-β1 gene-activated scaffold materials were found significantly higher than those of the control group （P〈0.05）. A brand-new way was provided for regulating seed cells to directionally differentiate into osteoblasts for bone defect restoration in bone tissue engineering.

Ethanol extract of Oenanthe javanica increases cell proliferation and neuroblast differentiation in the adolescent rat dentate gyrus

Oenanthe javanica is an aquatic perennial herb that belongs to theOenanthe genus in Apiaceae family, and it displays well-known medicinal properties such as protective effects against glu-tamate-induced neurotoxicity. However, few studies regarding effects ofOenanthe javanica on neurogenesis in the brain have been reported. In this study, we examined the effects of a normal diet and a diet containing ethanol extract ofOenanthe javanica on cell proliferation and neu-roblast differentiation in the subgranular zone of the hippocampal dentate gyrus of adolescent rats using Ki-67 （an endogenous marker for cell proliferation） and doublecortin （a marker for neuroblast）. Our results showed thatOenanthe javanica extract signiifcantly increased the number of Ki-67-immunoreactive cells and doublecortin-immunoreactive neuroblasts in the subgranular zone of the dentate gyrus in the adolescent rats. In addition, the immunoreactivity of brain-derived neurotrophic factor was signiifcantly increased in the dentate gyrus of the Oenanthe javanica extract-treated group compared with the control group. However, we did not ifnd that vascular endothelial growth factor expression was increased in theOenanthe javanica extract-treated group compared with the control group. These results indicate thatOenanthe javanica extract improves cell proliferation and neuroblast differentiation by increasing brain-de-rived neurotrophic factor immunoreactivity in the rat dentate gyrus.

Engineering vascularized organotypic tissues via module assembly

Adequate vascularization is a critical determinant for the successful construction and clinical implementation of complex organotypic tissue models. Currently, low cell and vessel density and insufficient vascular maturation make vascularized organotypic tissue construction difficult,greatly limiting its use in tissue engineering and regenerative medicine. To address these limitations, recent studies have adopted pre-vascularized microtissue assembly for the rapid generation of functional tissue analogs with dense vascular networks and high cell density. In this article, we summarize the development of module assembly-based vascularized organotypic tissue construction and its application in tissue repair and regeneration, organ-scale tissue biomanufacturing, as well as advanced tissue modeling.