Tissue-Engineered Regeneration of Completely Transected Spinal Cord Using Induced Neural Stem Cells and Gelatin-Electrospun Poly (Lactide-Co-Glycolide)/Polyethylene Glycol Scaffolds

Tissue engineering has brought new possibilities for the treatment of spinal cord injury. Two important components for tissue engineering of the spinal cord include a suitable cell source and scaffold. In our study, we investigated induced mouse embryonic fibroblasts (MEFs) directly reprogrammed into neural stem cells (iNSCs), as a cell source. Three-dimensional (3D) electrospun poly (lactide-co-glycolide)/polyethylene glycol (PLGA-PEG) nanofiber scaffolds were used for iNSCs adhesion and growth. Cell growth, survival and proliferation on the scaffolds were investigated. Scanning electron microcopy (SEM) and nuclei staining were used to assess cell growth on the scaffolds. Scaffolds with iNSCs were then transplanted into transected rat spinal cords. Two or 8 weeks following transplantation, immunofluorescence was performed to determine iNSC survival and differentiation within the scaffolds. Functional recovery was assessed using the Basso, Beattie, Bresnahan (BBB) Scale. Results indicated that iNSCs showed similar morphological features with wild-type neural stem cells (wt-NSCs), and expressed a variety of neural stem cell marker genes. Furthermore, iNSCs were shown to survive, with the ability to self-renew and undergo neural differentiation into neurons and glial cells within the 3D scaffolds in vivo. The iNSC-seeded scaffolds restored the continuity of the spinal cord and reduced cavity formation. Additionally, iNSC-seeded scaffolds contributed to functional recovery of the spinal cord. Therefore, PLGA-PEG scaffolds seeded with iNSCs may serve as promising supporting transplants for repairing spinal cord injury (SCI).     

Stem and progenitor cells for neurological repair: minor issues, major hurdles, and exciting opportunities for paracrine-based therapeutics

The transplantation of cultured stem and progenitor cells is a key element in the rapidly growing field of regenerative medicine. Based on their ability to rescue and/or repair injured tissue and partially restore organ function, multiple types of stem/progenitor cells have already entered into clinical trials. However, despite several decades of intense research, the goal to apply culture-expanded stem/progenitor cells in a manner that can effectively replace cells after injury has yet to be realized. Many sources of potentially useful cells are available, but something is clearly missing. In addition, recent studies suggest that paracrine effects of secreted or released factors are responsible for most of the benefits observed after cell transplantation, rather than direct cell replacement. These data call into question the need for cell transplantation for many types of therapy, in particular for acute injuries such as myocardial infarction and stroke. In this review, we examine current progress in the area of cell transplantation and minor issues and major hurdles regarding the clinical application of different cell types. We discuss the "paracrine hypothesis" for the action of transplanted stem/progenitor cells as an opportunity to identify defined combinations of biomolecules to rescue and/or repair tissues after injury. Although many of the concepts in this review will apply to multiple injury/repair systems, we will focus primarily on stem/progenitor cell-based treatments for neurological disorders and stroke.     

Neural stem cells, tumour stem cells and brain tumours: dangerous relationships?

Neural stem cells (NSC) have been implicated not only in brain development and neurogenesis but also in tumourigenesis. Brain tumour stem cells (BTSC) have been isolated from several paediatric or adult human brain tumours, however their origin is still disputed. This review discusses the normal role of NSC in the adult mammalian brain and their anatomical location. It compares the molecular characteristics and the biological behaviour of NSC/BTSC, and describes the molecular pathways involved in controlling self-renewal and maintenance of adult NSC/BTSC and brain tumour development. It also assesses the current hypotheses about the origin of BTSC and the clinical consequences.     

LncRNA SNHG6 functions as a ceRNA to regulate neuronal cell apoptosis by modulating miR‐181c‐5p/BIM signalling in ischaemic stroke

Long non‐coding RNAs (lncRNAs) play important roles in the pathogenesis of brain and neurodegenerative disorders. As far as we know, the functions and potential mechanisms of small nucleolar RNA host gene 6 (SNHG6) in ischaemic stroke have not been explored. This study aimed to examine the functional role of SNHG6 in the ischaemic stroke. Middle cerebral artery occlusion (MCAO) in mice and the oxygen glucose deprivation (OGD)‐induced injury in neuronal cells were applied to mimic ischaemic stroke. TTC staining, quantitative real‐time PCR, cell apoptosis assay, caspase‐3 activity assay, Western blot, RNA immunoprecipitation and luciferase reporter assay were performed to evaluate the function and possible mechanisms of SNHG6 in the pathogenesis of ischaemic stroke. The results show that SNHG6 expression was significantly increased both OGD‐induced neuronal cells and MCAO model mice. In vitro results showed that inhibition of SNHG6 increased cell viability, inhibited cell apoptosis and caspase‐3 activity in OGD‐induced neuronal cells. Consistently, knockdown of SNHG6 reduced brain infarct size and improved neurological scores in the MCAO mice. Mechanistic study further revealed that SNHG6 functioned as a competing endogenous RNA (ceRNA) for miR‐181c‐5p, which in turn repressed its downstream target of Bcl‐2 interacting mediator of cell death (BIM) and inhibiting cell apoptosis. This study revealed a novel function of SNHG6 in the modulating neuronal apoptosis in the ischaemic stroke model, and the role of SNHG6 in the regulating of neuronal apoptosis was at least partly via targeting miR‐181c‐5p/BIM signalling pathway.     

Direct reprogramming of Sertoli cells into multipotent neural stem cells by defined factors

Multipotent neural stem/progenitor cells hold great promise for cell therapy. The reprogramming of fibroblasts to induced pluripotent stem cells as well as mature neurons suggests a possibility to convert a terminally differentiated somatic cell into a multipotent state without first establishing pluripotency. Here, we demonstrate that Sertoli cells derived from mesoderm can be directly converted into a multipotent state that possesses neural stem/progenitor cell properties. The induced neural stem/progenitor cells (iNSCs) express multiple NSC-specific markers, exhibit a global gene-expression profile similar to normal NSCs, and are capable of self-renewal and differentiating into glia and electrophysiologically functional neurons. iNSC-derived neurons stain positive for tyrosine hydroxylase (TH), γ-aminobutyric acid, and choline acetyltransferase. In addition, iNSCs can survive and generate synapses following transplantation into the dentate gyrus. Generation of iNSCs may have important implications for disease modeling and regenerative medicine.     

Embryonic stem cell-derived L1 overexpressing neural aggregates enhance recovery after spinal cord injury in mice

An obstacle to early stem cell transplantation into the acutely injured spinal cord is poor survival of transplanted cells. Transplantation of embryonic stem cells as substrate adherent embryonic stem cell-derived neural aggregates (SENAs) consisting mainly of neurons and radial glial cells has been shown to enhance survival of grafted cells in the injured mouse brain. In the attempt to promote the beneficial function of these SENAs, murine embryonic stem cells constitutively overexpressing the neural cell adhesion molecule L1 which favors axonal growth and survival of grafted and imperiled cells in the inhibitory environment of the adult mammalian central nervous system were differentiated into SENAs and transplanted into the spinal cord three days after compression lesion. Mice transplanted with L1 overexpressing SENAs showed improved locomotor function when compared to mice injected with wild-type SENAs. L1 overexpressing SENAs showed an increased number of surviving cells, enhanced neuronal differentiation and reduced glial differentiation after transplantation when compared to SENAs not engineered to overexpress L1. Furthermore, L1 overexpressing SENAs rescued imperiled host motoneurons and parvalbumin-positive interneurons and increased numbers of catecholaminergic nerve fibers distal to the lesion. In addition to encouraging the use of embryonic stem cells for early therapy after spinal cord injury L1 overexpression in the microenvironment of the lesioned spinal cord is a novel finding in its functions that would make it more attractive for pre-clinical studies in spinal cord regeneration and most likely other diseases of the nervous system.     

A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or α-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.     

神经干细胞移植的临床研究进展

神经系统损伤会导致脑内神经干细胞（neural stem cells,NSCs）的扩增以实现自我修复功能,而通过外源细胞移植的方式来加速这一进程,可能是一种更有效的治疗手段。当前,神经干细胞临床研究所面临的主要问题是如何评价细胞在移植后的行为和功能。该文综述了近几年使用神经干细胞移植治疗几种主要神经系统疾病的临床研究成果,并着重关注了干细胞移植后的示踪研究。     

胚胎干细胞的心脏应用

心肌梗死期间死亡的心肌细胞将由没有收缩功能的疤痕组织替代,因而极可能引起心力衰竭。对治疗心衰来说,修复死亡或损伤的心肌以及改善心功能仍面临着极大挑战。干细胞移植已在近年来的实验中用于修复损失的心肌。本文总结了近期在心肌损伤动物中实施胚胎干细胞移植的实验结果,并着重介绍对这类特定细胞的研究进展。胚胎干细胞取源于早期哺乳类胚胎的胚芽细胞,属于多功能干细胞。这类细胞具有长期增殖而不分化的能力,或台色够在培养过程中分化成包括心肌细胞在内的所有特殊体细胞。由于胚胎干细胞具有极大的增殖和分化为成熟组织的能力,它们可能成为一种潜在的很有实用价值的细胞来源,可用于对病态心脏的功能心肌再生的细胞治疗。新近的研究表明,在心肌梗死动物模型中,心肌内移植胚胎干细胞或由其分化成的心肌样细胞,能导致已损伤心肌的再生,并改善心脏功能。另外,在病毒性心肌炎小鼠中,静脉输入胚胎干细胞可明显提高生存率和减轻心肌损伤。有关人类胚胎干细胞在体外分化成心肌细胞以及这些细胞的特性,近来已有报道。然而,要在临床能应用人类胚胎干细胞或由其分化成的心肌细胞来治疗晚期心脏疾病,还必须越过大量的伦理、法律和科学上的障碍。     

Long-Term Extensive Ectopic Hair Growth on the Spinal Cord of Mice from Transplanted Whisker Follicles

We have previously demonstrated that hair follicles contain nestin-expressing pluripotent stem cells that can effect nerve and spinal cord repair upon transplantation. In the present study, isolated whisker follicles from nestin-driven green fluorescent protein (ND-GFP) mice were histocultured on Gelfoam for 3 weeks for the purpose of transplantation to the spinal cord to heal an induced injury. The hair shaft was cut off from Gelfoam-histocultured whisker follicles, and the remaining part of the whisker follicles containing GFP-nestin expressing pluripotent stem cells were transplanted into the injured spinal cord of nude mice, along with the Gelfoam. After 90 days, the mice were sacrificed and the spinal cord lesion was observed to have healed. ND-GFP expression was intense at the healed area of the spinal cord, as observed by fluorescence microscopy, demonstrating that the hair follicle stem cells were involved in healing the spinal cord. Unexpectedly, the transplanted whisker follicles sprouted out remarkably long hair shafts in the spinal cord during the 90 days after transplantation of Gelfoam whisker histocultures to the injured spine. The pigmented hair fibers, grown from the transplanted whisker histocultures, curved and enclosed the spinal cord. The unanticipated results demonstrate the great potential of hair growth after transplantation of Gelfoam hair follicle histocultures, even at an ectopic site.     

Mesenchymal stem cells showed the highest potential for the regeneration of injured liver tissue compared with other subpopulations of the bone marrow

We have previously reported that bone marrow cells (BMCs) participate in the regeneration after liver injury. However, it is not established that this is the result of differentiation of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) or the combination of both. We investigated the contribution of each cell fraction to the regenerative process. First, we confirmed that transplanted stem cells migrate directly to injured liver tissue without dispersing to other organs. Next, we divided green fluorescent protein (GFP)-expressing BMCs into three populations as mononuclear cells, MSCs and HSCs. We then compared the engraftment capacity after transplantation of each fraction of cells into liver-injured mice. Of these, the MSCs transplanted group showed the highest GFP fluorescence intensities in liver tissue by flow cytometry analysis and confocal microscopic observation. Furthermore, MSCs showed differentiation potential into hepatocytes when co-cultured with injured liver cells, which suggests that MSCs showed highest potential for the regeneration of injured liver tissue compared with those of the other two cell refractions.     

Transdifferentiation-Induced Neural Stem Cells Promote Recovery of Middle Cerebral Artery Stroke Rats

Induced neural stem cells (iNSCs) can be directly transdifferentiated from somatic cells. One potential clinical application of the iNSCs is for nerve regeneration. However, it is unknown whether iNSCs function in disease models. We produced transdifferentiated iNSCs by conditional overexpressing Oct4, Sox2, Klf4, c-Mycin mouse embryonic fibroblasts. They expanded readily in vitro and expressed NSC mRNA profile and protein markers. These iNSCs differentiated into mature astrocytes, neurons and oligodendrocytes in vitro. Importantly, they reduced lesion size, promoted the recovery of motor and sensory function as well as metabolism status in middle cerebral artery stroke rats. These iNSCs secreted nerve growth factors, which was associated with observed protection of neurons from apoptosis. Furthermore, iNSCs migrated to and passed through the lesion in the cerebral cortex, where Tuj1+ neurons were detected. These findings have revealed the function of transdifferentiated iNSCs in vivo, and thus provide experimental evidence to support the development of personalized regenerative therapy for CNS diseases by using genetically engineered autologous somatic cells.     

Therapeutic Use of Stem Cell Transplantation for Cell Replacement or Cytoprotective Effect of Microvesicle Released from Mesenchymal Stem Cell

Idiopathic pulmonary fibrosis (IPF) is the most common and severe type of idiopathic interstitial pneumonias (IIP), and which is currently no method was developed to restore normal structure and function. There are several reports on therapeutic effects of adult stem cell transplantations in animal models of pulmonary fibrosis. However, little is known about how mesenchymal stem cell (MSC) can repair the IPF. In this study, we try to provide the evidence to show that transplanted mesenchymal stem cells directly replace fibrosis with normal lung cells using IPF model mice. As results, transplanted MSC successfully integrated and differentiated into type II lung cell which express surfactant protein. In the other hand, we examine the therapeutic effects of microvesicle treatment, which were released from mesenchymal stem cells. Though the therapeutic effects of MV treatment is less than that of MSC treatment, MV treat-ment meaningfully reduced the symptom of IPF, such as collagen deposition and inflammation. These data suggest that stem cell transplantation may be an effective strategy for the treatment of pulmonary fibrosis via replacement and cytoprotective effect of microvesicle released from MSCs.     

Direct transplantation of uncultured hair‐follicle pluripotent stem (hfPS) cells promotes the recovery of peripheral nerve injury

We previously showed that the stem cell marker nestin is expressed in hair follicle stem cells which suggested their pluripotency. We subsequently showed that the nestin‐expressing hair‐follicle pluripotent stem (hfPS) cells can differentiate in culture to neurons, glial cells, keratinocytes, and other cell types and can promote regeneration of peripheral nerve and spinal cord injuries upon injection to the injured nerve or spinal cord. The location of the hfPS cells has been termed the hfPS cell area (hfPSCA). Previously, hfPS cells were cultured for 1–2 months before transplantation to the injured nerve or spinal cord which would not be optimal for clinical application of these cells for nerve or spinal cord repair, since the patient should be treated soon after injury. In the present study, we addressed this issue by directly using the upper part of the hair follicle containing the hfPSCA, without culture, for injection into the severed sciatic nerve in mice. After injection of hfPSCA, the implanted hfPS cells grew and promoted joining of the severed nerve. The transplanted hfPS cells differentiated mostly to glial cells forming myelin sheaths, which promoted axonal growth and functional recovery of the severed nerve. These results suggest that the direct transplantation of the uncultured upper part of the hair follicle containing the hfPSA is an important method to promote the recovery of peripheral nerve injuries and has significant clinical potential. J. Cell. Biochem. 110: 272–277, 2010. © 2010 Wiley‐Liss, Inc.     

Bone marrow mesenchymal stem cells can differentiate and assume corneal keratocyte phenotype

It remains elusive as to what bone marrow (BM) cell types infiltrate into injured and/or diseased tissues and subsequently differentiate to assume the phenotype of residential cells, for example, neurons, cardiac myocytes, keratocytes, etc., to repair damaged tissue. Here, we examined the possibility of whether BM cell invasion via circulation into uninjured and injured corneas could assume a keratocyte phenotype, using chimeric mice generated by transplantation of enhanced green fluorescent protein (EGFP)(+) BM cells into keratocan null (Kera(-/-)) and lumican null (Lum(-/-)) mice. EGFP(+) BM cells assumed dendritic cell morphology, but failed to synthesize corneal-specific keratan sulfate proteoglycans, that is KS-lumican and KS-keratocan. In contrast, some EGFP(+) BM cells introduced by intrastromal transplantation assumed keratocyte phenotypes. Furthermore, BM cells were isolated from Kera-Cre/ZEG mice, a double transgenic mouse line in which cells expressing keratocan become EGFP(+) due to the synthesis of Cre driven by keratocan promoter. Three days after corneal and conjunctival transplantations of such BM cells into Kera(-/-) mice, green keratocan positive cells were found in the cornea, but not in conjunctiva. It is worthy to note that transplanted BM cells were rejected in 4 weeks. MSC isolated from BM were used to examine if BM mesenchymal stem cells (BM-MSC) could assume keratocyte phenotype. When BM-MSC were intrastromal-transplanted into Kera(-/-) mice, they survived in the cornea without any immune and inflammatory responses and expressed keratocan in Kera(-/-) mice. These observations suggest that corneal intrastromal transplantation of BM-MSC may be an effective treatment regimen for corneal diseases involving dysfunction of keratocytes.     

成体心脏干细胞经静脉移植后在心梗小鼠体内的分布研究

目的：观察心肌梗死小鼠静脉移植成体心脏干细胞后,细胞在小鼠体内各器官的分布情况。明确心梗后静脉移植成体心脏干 细胞在小鼠体内的分布和归巢情况。方法：分离培养小鼠心脏成体干细胞,采用流式细胞仪鉴定细胞,通过亲脂性染料CM-DiI标 记细胞后行小鼠急性心肌梗死模型建立和细胞移植,分别在细胞移植后7、14、8 天取小鼠心脏、肝脏、脑、脊髓、肺脏,行冰冻切 片,在荧光显微镜下观察移植细胞在各组织器官存活和分布情况。结果：成体心脏干细胞分离培养后呈贴壁生长,流式细胞仪检 测显示细胞纯度>80%。CM-DiI标记后荧光显微镜下观察可见标记的细胞胞浆胞核均被染成呈明亮的红色。心肌梗死后经静脉 移植成体心脏干细胞,细胞在各组织中分布呈变化过程,7 天时,在肺脏和肝脏分布较多,至14 天和28 天时,肺脏和肝脏分布减 少,心脏分布逐渐增多,表现出向心脏的" 归巢" 现象,而脑和脊髓在28 天的观察时间内分布较少。结论：采用CM-DiI标记心 脏成体干细胞,操作简单,标记效果好,可用于短期的细胞体内追踪。小鼠心肌梗死后行经静脉成体心脏干细胞移植,28 天后细胞 在心脏的分布逐渐增多,表现出向心脏的" 归巢" 现象。     

Human umbilical cord blood cells improve cardiac function after myocardial infarction

Human umbilical cord blood (UCB) contains an abundance of immature stem/progenitor cells and has been clinically used as an alternative to bone marrow transplantation. In addition, cord blood can be obtained non-invasively, in contrast to invasive bone marrow aspiration. We investigated the potential of human UCB CD34(+) cells to improve cardiac function following myocardial infarction. Myocardial infarction was induced in Wistar rats by ligation of the left coronary artery. Either 2x10(5) human UCB CD34(+) cells or equivalent cell-free medium was injected into the injured myocardium of the rats following induction of myocardial infarction. CD34(+) cell transplantation significantly improved ventricular function as compared to the control group. Immunofluorescence staining for human CD34, CD45, and PECAM-1 revealed surviving cells in the myocardium. Our findings suggest that transplanted human cells survived and improved cardiac function following myocardial infarction. These results may show the usefulness of UCB CD34(+) cells for myocardial infarction.