Knockdown of IKK1/2 Promotes Differentiation of Mouse Embryonic Stem Cells into Neuroectoderm at the Expense of Mesoderm

Activation of nuclear factor kappa B (NF-??B) is accomplished by a specific kinase complex (IKK-complex), phosphorylating inhibitors of NF-??B (I??B). In embryonic stem cells (ESCs), NF-??B signaling causes loss of pluripotency and promotes differentiation towards a mesodermal phenotype. Here we show that NF-??B signaling is involved in cell fate determination during retinoic acid (RA) mediated differentiation of ESCs. Knockdown of IKK1 and IKK2 promotes differentiation of ESCs into neuroectoderm at the expense of neural crest derived myofibroblasts. Our data indicate that RA is not only able to induce neuronal differentiation in vitro but also drives ESCs into a neural crest cell lineage represented by differentiation towards peripheral neurons and myofibroblasts. The NC is a transiently existing, highly multipotent embryonic cell population generating a wide range of different cell types. During embryonic development the NC gives rise to distinct precursor lineages along the anterior-posterior axis determining differentiation towards specific derivates. Retinoic acid (RA) signaling provides essential instructive cues for patterning the neuroectoderm along the anterior-posterior axis. The demonstration of RA as a sufficient instructive signal for the differentiation of pluripotent cells towards NC and the involvement of NF-??B during this process provides useful information for the generation of specific NC-lineages, which are valuable for studying NC development or disease modeling.     

Collagen microgel-assisted dexamethasone release from PLLA-collagen hybrid scaffolds of controlled pore structure for osteogenic differentiation of mesenchymal stem cells

Directed stem cell differentiation over three-dimensional porous scaffolds capable of releasing bioactive instructive cues is an important tool in tissue engineering. In this research, we have prepared dexamethasone (Dex)-releasing collagen microbead-functionalized poly(L-Lactide)-collagen hybrid scaffolds as an osteoinductive platform for human bone marrow-derived mesenchymal stem cells (MSCs). The scaffolds were prepared by a combined method of emulsion freeze-drying and porogen-leaching using pre-prepared ice collagen particulates as a porogen material. Dex release from the hybrid scaffolds was studied at 37?°C under shaking condition and the impact of released Dex towards osteogenic lineage differentiation was investigated by 3?week in vitro culture of MSCs. The results showed that hybrid scaffolds had controlled pore structure and interconnected pores deposited with collagen fibers. The hybrid scaffold facilitated cell seeding and the spatial localization of Dex/collagen microbeads facilitated a microgel-assisted spatio-temporal control of Dex release. The released Dex was useful for osteogenic differentiation of MSCs, which was confirmed from the elevated expression of osteogenic-specific gene-encoded proteins. The hybrid scaffolds should be useful for regeneration of a functional bone tissue.     

自体外周血干细胞移植治疗周围神经损伤

背景：关于神经干细胞对周围神经损伤的治疗已有多篇报道,但外周血干细胞对周围神经损伤治疗鲜有报道。 目的：探讨自体外周血干细胞移植治疗周围神经损伤使失神经骨骼肌重获神经再支配的临床应用。 方法：应用外周血干细胞治疗周围神经损伤6例,同时与周围神经损伤单纯行神经断端吻合或神经移植10例比较。2组患者术后常规肌注鼠神经生长因子一两个疗程,同时给予针灸、理疗、经皮电刺激治疗及功能康复训练。 结果与结论：两组患者随访均超过6个月。干细胞移植组运动神经传导速度和感觉神经传导速度的恢复率要明显高于单纯神经吻合组。提示周围神经损伤后给予修复局部用外周血干细胞移植能够使远端失神经骨骼肌早期重新获得神经再支配。     

控释神经营养因子与细胞移植减少损伤脊髓的胶质瘢痕

背景：前期研究发现控释胶质细胞源性神经营养因子与骨髓间充质干细胞源神经元样细胞联合移植可有效促进猕猴脊髓损伤后运动功能和感觉功能的恢复。 目的：观察控释胶质细胞源性神经营养因子联合骨髓间充质干细胞源神经元样细胞移植抑制猴脊髓损伤后胶质瘢痕形成的作用是否优于单纯细胞移植。 方法：取12只恒河猴,采用改良Allen氏法制作急性重度脊髓损伤模型,随机数字表法分为3组,实验组以控释胶质细胞源性神经营养因子联合自体骨髓间充质干细胞源神经元样细胞移植修复,对照组以自体骨髓间充质干细胞源神经元样细胞移植修复,空白对照组以磷酸盐缓冲液修复。修复后5个月,取出脊髓组织制成石蜡标本,应用免疫组织化学染色显示胶质瘢痕的形态特征、构成特点及瘢痕中神经纤维的再生情况,检测胶质瘢痕面积及胶质纤维酸性蛋白染色的平均吸光度值。 结果与结论：脊髓损伤部位胶质瘢痕由混合性增生的星形胶质细胞和组织细胞构成。空白对照组脊髓胶质瘢痕累及范围广,星形胶质细胞增生显著,神经丝蛋白免疫组织化学染色阴性,胶质瘢痕面积、胶质纤维酸性蛋白染色平均吸光度值高于实验组与对照组(P < 0.05);实验组、对照组脊髓胶质瘢痕累及范围较局限,神经丝蛋白免疫组织化学染色显示有少量神经纤维通过瘢痕区,并且实验组胶质瘢痕面积、胶质纤维酸性蛋白染色平均吸光度值低于对照组(P < 0.05)。结果表明,控释胶质细胞源性神经营养因子联合骨髓间充质干细胞源性神经元样细胞移植可更强抑制脊髓损伤后胶质瘢痕的形成。     

经血源性子宫内膜干细胞及其修复外周神经损伤的应用前景

外周神经损伤带给患者长期病痛的同时严重影响了患者的生活质量,目前临床上除显微外科手术外,移植施万细胞(SC)也成为有效的治疗方法。然而SC移植的先天不足限制了其在临床上的大规模使用,因此,干细胞治疗神经损伤逐渐成为近年来的研究热点。而近来发现的经血源性子宫内膜干细胞(Men ESCs)凭借其丰富的来源,无创伤的分离方式以及较高的增殖活性和分化潜能等方面的优势获得广泛关注,并在成骨、心肌、肝脏、子宫内膜及中风等疾病的治疗过程中显示了良好的效果。因此,我们在明确外周神经损伤发生机制的基础上,着重阐明Men ESCs的生物学特性及其在治疗外周神经损伤的机制,以期为Men ESCs在外周神经损伤的治疗提供借鉴。     

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.     

依达拉奉联合神经干细胞移植修复大鼠脊髓损伤

背景：依达拉奉是一种自由基清除药,可以减轻受损神经组织水肿和改善脊髓损伤区微环境。 目的：观察依达拉奉联合神经干细胞移植对大鼠脊髓全横断损伤的修复效果。 方法：成年雌性SD大鼠80只,建立胸9脊髓全横断损伤模型,随机分为4组：对照组不做处理;依达拉奉组脊髓损伤后6 h经尾静脉注射依达拉奉;神经干细胞移植组脊髓损伤后6 h脊髓损伤区域注入神经干细胞悬液;依达拉奉+细胞移植组脊髓损伤后6 h神经干细胞移植的同时尾静脉注射依达拉奉。 结果与结论：造模后8周可观察到PKH-26标记的神经干细胞在体内存活并在脊髓内迁移;细胞移植组和依达拉奉联+细胞移植组可见少量连续性神经纤维通过损伤区。荧光金逆行脊髓追踪显示神经干细胞移植组和依达拉奉+细胞移植组可见被荧光金标记的神经锥体细胞穿越损伤区。PKH-26标记的阳性细胞数及荧光金阳性神经纤维数：依达拉奉+细胞移植组最多,依达拉奉组、神经干细胞移植组次之,对照组最少,各组之间差异有显著性意义(P < 0.05);后肢功能运动BBB评分依次为依达拉奉+细胞移植组>神经干细胞移植组>依达拉奉组>对照组。提示依达拉奉能促进神经干细胞在损伤区的存活并向神经细胞分化,依达拉奉联合神经干细胞移植有促进细胞移植修复大鼠脊髓损伤的效果。     

Erk1/2 promotes proliferation and inhibits neuronal differentiation of neural stem cells

Neural stem cells (NSCs) are in a complex niche in which cell-extrinsic cues and cell-intrinsic genetic mechanisms in chorus mediate their cellular processes such as self-renewal and differentiation. In this study, we found that inactivation of Erk1/2 with U0126 in NSCs significantly promoted neuronal differentiation and inhibited proliferation. Sustained Erk1/2 inactivity was required in this process. We also found that nerve growth factor (NGF) and collagen could promote the proliferation and inhibit neuronal differentiation by activating phosphorylation of Erk1/2. Cell-cycle regulators such as cyclin-dependent kinase 2 (Cdk2), Cyclin D1 and Hes1 mediated the effect of Erk on NSCs proliferation and differentiation. Our results showed that Erk1/2 played an important role in the interplay between cell-extrinsic cues and cell-intrinsic genetic mechanisms in neural stem cell biology.     

神经干细胞移植与脑梗死

背景：将神经干细胞移植到脑梗死病灶区可以促进受损神经细胞的修复。 目的：分析神经干细胞移植治疗脑梗死的相关影响因素。 方法：文章通过数据库检索的方式分析近年来关于神经干细胞移植治疗脑梗死的实验研究,从中国临床试验注册和基础实验研究两方面分析神经干细胞与脑梗死的研究进展。 结果与结论：脑梗死后神经干细胞的增殖和分化与脑内微环境密切相关,梗死区域有大量的神经细胞丢失。细胞因子在神经干细胞移植修复神经功能损伤时发挥一定作用,能够诱导大鼠脑梗死后神经干细胞的增殖、分化和迁移,目前被证实的有表皮生长因子、脑源性神经营养因子、胰岛素样生长因子1、神经生长因子、碱性成纤维细胞生长因子等。针灸和中医药对脑梗死后室管膜下区神经干细胞的增殖、迁移和分化有促进作用。神经干细胞移植治疗脑梗死的研究已经取得了阶段性进展,未来仍有许多问题需要进一步解决。     

Spatial control of adult stem cell fate using nanotopographic cues

Adult stem cells hold great promise as a source of diverse terminally differentiated cell types for tissue engineering applications. However, due to the complexity of chemical and mechanical cues specifying differentiation outcomes, development of arbitrarily complex geometric and structural arrangements of cells, adopting multiple fates from the same initial stem cell population, has been difficult. Here, we show that the topography of the cell adhesion substratum can be an instructive cue to adult stem cells and topographical variations can strongly bias the differentiation outcome of the cells towards adipocyte or osteocyte fates. Switches in cell fate decision from adipogenic to osteogenic lineages were accompanied by changes in cytoskeletal stiffness, spanning a considerable range in the cell softness/rigidity spectrum. Our findings suggest that human mesenchymal stem cells (hMSC) can respond to the varying density of nanotopographical cues by regulating their internal cytoskeletal network and use these mechanical changes to guide them toward making cell fate decisions. We used this finding to design a complex two-dimensional pattern of co-localized cells preferentially adopting two alternative fates, thus paving the road for designing and building more complex tissue constructs with diverse biomedical applications.     

维甲酸、音猬因子对大鼠脊髓神经干细胞向运动神经元样细胞分化的影响

目的:分离大鼠胚胎的脊髓神经干细胞进行体外培养,探讨维甲酸(RA)、音猬因子(Shh)诱导其向运动神经元样细胞分化. 方法:应用无血清培养技术分离培养脊髓神经干细胞,通过5-溴-2-脱氧尿苷标记、免疫荧光显色检测细胞增殖、分化情况;培养液分组添加Shh、 RA或Shh+RA,观察神经干细胞向运动神经元样细胞的分化情况. 结果:大鼠胚胎脊髓可成功分离神经干细胞,分化后可表达神经元、星形胶质细胞的特异性抗原;诱导分化后结果显示Shh组未检测到胆碱乙酰转移酶阳性细胞,RA组为20.63%, Shh+RA组为66.84%,其差异具统计学意义.结论:从大鼠胚胎脊髓可成功分离神经干细胞,Shh、 RA可不同程度地诱导脊髓神经干细胞分化为运动神经元样细胞.     

Bioprinting of growth factors onto aligned sub-micron fibrous scaffolds for simultaneous control of cell differentiation and alignment

The capability to spatially control stem cell orientation and differentiation simultaneously using a combination of geometric cues that mimic structural aspects of native extracellular matrix (ECM) and biochemical cues such as ECM-bound growth factors (GFs) is important for understanding the organization and function of musculoskeletal tissues. Herein, oriented sub-micron fibers, which are morphologically similar to musculoskeletal ECM, were spatially patterned with GFs using an inkjet-based bioprinter to create geometric and biochemical cues that direct musculoskeletal cell alignment and differentiation in vitro in registration with fiber orientation and printed patterns, respectively. Sub-micron polystyrene fibers (diameter ~ 655 nm) were fabricated using a Spinneret-based Tunable Engineered Parameters (STEP) technique and coated with serum or fibrin. The fibers were subsequently patterned with tendon-promoting fibroblast growth factor-2 (FGF-2) or bone-promoting bone morphogenetic protein-2 (BMP-2) prior to seeding with mouse C2C12 myoblasts or C3H10T1/2 mesenchymal fibroblasts. Unprinted regions of STEP fibers showed myocyte differentiation while printed FGF-2 and BMP-2 patterns promoted tenocyte and osteoblast fates, respectively, and inhibited myocyte differentiation. Additionally, cells aligned along the fiber length. Functionalizing oriented sub-micron fibers with printed GFs provides instructive cues to spatially control cell fate and alignment to mimic native tissue organization and may have applications in regenerative medicine.     

Induced pluripotent stem cells for neural tissue engineering

Induced pluripotent stem cells (iPSCs) hold great promise for cell therapies and tissue engineering. Neural crest stem cells (NCSCs) are multipotent and represent a valuable system to investigate iPSC differentiation and therapeutic potential. Here we derived NCSCs from human iPSCs and embryonic stem cells (ESCs), and investigated the potential of NCSCs for neural tissue engineering. The differentiation of iPSCs and the expansion of derived NCSCs varied in different cell lines, but all NCSC lines were capable of differentiating into mesodermal and ectodermal lineages, including neural cells. Tissue-engineered nerve conduits were fabricated by seeding NCSCs into nanofibrous tubular scaffolds, and used as a bridge for transected sciatic nerves in a rat model. Electrophysiological analysis showed that only NCSC-engrafted nerve conduits resulted in an accelerated regeneration of sciatic nerves at 1 month. Histological analysis demonstrated that NCSC transplantation promoted axonal myelination. Furthermore, NCSCs differentiated into Schwann cells and were integrated into the myelin sheath around axons. No teratoma formation was observed for up to 1 year after NCSC transplantation in vivo. This study demonstrates that iPSC-derived multipotent NCSCs can be directly used for tissue engineering and that the approach that combines stem cells and scaffolds has tremendous potential for regenerative medicine applications.     

Single neural progenitor cells derived from EGFP expressing mice is useful after spinal cord injury in mice

Neural stem cells (NSCs) were widely used for studying the cell's replacement after transplantation in nervous system because of its specific characteristics. However, Stracing the cells after transplantation was still a problem. In the present study, we isolated and cultured the neural stem cells from the C57BL/6J EGFP transgenic mouse (EGFP mice), and identified the capacity for self-renewal and differentiation into the three CNS lineages (neurons, astrocytes, and oligodendrocytes). Then we transplanted the single neural stem cell into the lesion spinal cord. Expression of GFP and differentiation was evaluated at two weeks post-transplantation. The data showed that these neural stem cells derived from the EGFP mice could maintain transgene expression and could differentiate into the MAP2 positive cells after transplantation into the injured spinal cord. The results suggested that NSC expressing EGFP was a useful marker for tracing the cells after transplantation in vivo and functional in the treatment to spinal cord injury.     

电针刺激对神经干细胞移植治疗脊髓损伤大鼠后肢功能的影响

背景：单纯的神经干细胞移植对受损脊髓组织的修复作用并不理想,为了进一步提高移植细胞在体内的存活、增殖及定向分化为神经元的比例,必须进一步改善脊髓损伤区的微环境。 目的：观察神经干细胞移植联合电针刺激对脊髓损伤大鼠后肢功能及电生理的影响。 方法：将脊髓损伤模型SD大鼠72只按随机数字表法分为4组：对照组尾静脉注入培养液,神经干细胞组经尾静脉注入等体积神经干细胞悬液,电针刺激组自模型完成6 h起采用督脉加体穴电针1周,联合组尾静脉注射神经干细胞后,同时采用督脉加体穴电针1周。分别于造模前、造模后1,3 d、1-4 周通过BBB评分、斜板试验进行运动功能评定。造模后4周取材行病理切片苏木精-伊红染色,荧光显微镜观测CM-Dil 标记的神经干细胞存活及分布情况,辣根过氧化物酶示踪观察神经纤维再生情况,运动诱发电位和体感诱发电位观察大鼠神经电生理恢复情况。 结果与结论：造模后2-4周大鼠下肢运动功能评价联合组优于神经干细胞组及电针刺激组,神经干细胞组和电针刺激组优于对照组。造模后4周,神经干细胞组和电针刺激组损伤区可见少量神经轴索样结构,脊髓空洞较小,联合组可见较多神经轴索样结构,未见脊髓空洞。造模后4周,CM-Dil 阳性细胞和辣根过氧化物酶阳性神经纤维数：联合组>神经干细胞组与电针刺激组>对照组,各组之间差异有显著性意义(P < 0.05)。运动诱发电位和体感诱发电位的潜伏期：联合组<神经干细胞组与电针刺激组<对照组,各组之间差异有显著性意义(P < 0.05);运动诱发电位和体感诱发电位的波幅：联合组>神经干细胞组与电针刺激组>对照组,各组之间差异有显著性意义(P < 0.05)。结果提示神经干细胞移植的同时联合电针刺激能够促进脊髓损伤大鼠神经突触的再生,改善其大鼠肢体运动功能及电生理功能。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程      

Histamine stimulates neurogenesis in the rodent subventricular zone

Neural stem/progenitor cells present in the subventricular zone (SVZ) are a potential source of repairing cells after injury. Therefore, the identification of novel players that modulate neural stem cells differentiation can have a huge impact in stem cell-based therapies. Herein, we describe a unique role of histamine in inducing functional neuronal differentiation from cultured mouse SVZ stem/progenitor cells. This proneurogenic effect depends on histamine 1 receptor activation and involves epigenetic modifications and increased expression of Mash1, Dlx2, and Ngn1 genes. Biocompatible poly (lactic-co-glycolic acid) microparticles, engineered to release histamine in a controlled and prolonged manner, also triggered robust neuronal differentiation in vitro. Preconditioning with histamine-loaded microparticles facilitated neuronal differentiation of SVZ-GFP cells grafted in hippocampal slices and in in vivo rodent brain. We propose that neuronal commitment triggered by histamine per se or released from biomaterial-derived vehicles may represent a new tool for brain repair strategies. STEM CELLS 2012; 30:773-784.     

Induction of mice adult bone marrow mesenchymal stem cells into functional motor neuron-like cells

The differentiation of mesenchymal stem cells (MSC) into acetylcholine secreted motor neuron-like cells, followed by elongation of the cell axon, is a promising treatment for spinal cord injury and motor neuron cell dysfunction in mammals. Differentiation is induced through a pre-induction step using Beta- mercaptoethanol (BME) followed by four days of induction with retinoic acid and sonic hedgehog. This process results in a very efficient differentiation of BM-MSCs into motor neuron-like cells. Immunocytochemistry showed that these treated cells had specific motor neural markers: microtubule associated protein-2 and acetylcholine transferase. The ability of these cells to function as motor neuron cells was assessed by measuring acetylcholine levels in a culture media during differentiation. High-performance liquid chromatography (HPLC) showed that the differentiated cells were functional. Motor neuron axon elongation was then induced by adding different concentrations of a nerve growth factor (NGF) to the differentiation media. Using a collagen matrix to mimic the natural condition of neural cells in a three-dimensional model showed that the MSCs were successfully differentiated into motor neuron-like cells. This process can efficiently differentiate MSCs into functional motor neurons that can be used for autologous nervous system therapy and especially for treating spinal cord injuries.     

The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation

Neural stem/progenitor cells (NSCs) are capable of self-renewal and differentiation into all types of neural lineage under different biochemical and topographical cues. In this study, we cultured rat hippocampus-derived adult NSCs (rNSCs) on laminin-coated electrospun Polyethersulfone (PES) fiber meshes with average fiber diameters of 283+/-45 nm, 749+/-153 nm and 1452+/-312 nm; and demonstrated that fiber diameter of PES mesh significantly influences rNSC differentiation and proliferation. Under the differentiation condition (in the presence of 1 microM retinoic acid and 1% fetal bovine serum), rNSCs showed a 40% increase in oligodendrocyte differentiation on 283-nm fibers and 20% increase in neuronal differentiation on 749-nm fibers, in comparison to tissue culture polystyrene surface. SEM imaging revealed that cells stretched multi-directionally to follow underlying 283-nm fibers, but extended along a single fiber axis on larger fibers. When cultured on fiber meshes in serum free medium in the presence of 20 ng/mL of FGF-2, rNSCs showed lower proliferation and more rounded morphology compared to that cultured on laminin-coated 2D surface. As the fiber diameter decreased, higher degree of proliferation and cell spreading and lower degree of cell aggregation were observed. This collective evidence indicates fiber topography can play a vital role in regulating differentiation and proliferation of rNSCs in culture.     

红景天苷对新生大鼠海马区神经干细胞分化的研究

目的研究红景天苷药物血清诱导新生大鼠海马神经干细胞向神经元方向分化,并促进所分化神经元细胞发育的作用。方法从新生24h内的Wistar大鼠脑中分离扩增获得大量神经干细胞后,加入低、中、高剂量的红景天苷药物血清及对照血清,观察其对神经干细胞分化为神经元的影响,并通过免疫细胞化学染色检测神经干细胞分化为神经元的状况。结果各药物实验组诱导神经干细胞分化为NSE阳性细胞的个数面积与周长明显高于对照组(P<0.05),分化为GFAP阳性细胞的个数明显低于对照组,且呈量效依赖关系。结论红景天苷药物学清在体外可促进神经干细胞向神经元方向分化,在一定范围内存在量效依赖关系,对所分化的神经元细胞有促生长发育作用。