Human neural stem cells promote proliferation of endogenous neural stem cells and enhance angiogenesis in ischemic rat brain

Ryu, Sun,Lee, Seung-Hoon,Kim, Seung U.,Yoon, Byung-Woo Medknow PublicationsMedia Pvt Ltd 2016 Neural regeneration research Vol.11 No.2

<P>Transplantation of human neural stem cells into the dentate gyrus or ventricle of rodents has been reportedly to enhance neurogenesis. In this study, we examined endogenous stem cell proliferation and angiogenesis in the ischemic rat brain after the transplantation of human neural stem cells. Focal cerebral ischemia in the rat brain was induced by middle cerebral artery occlusion. Human neural stem cells were transplanted into the subventricular zone. The behavioral performance of human neural stem cells-treated ischemic rats was significantly improved and cerebral infarct volumes were reduced compared to those in untreated animals. Numerous transplanted human neural stem cells were alive and preferentially localized to the ipsilateral ischemic hemisphere. Furthermore, 5-bromo-2′-deoxyuridine-labeled endogenous neural stem cells were observed in the subventricular zone and hippocampus, where they differentiated into cells immunoreactive for the neural markers doublecortin, neuronal nuclear antigen NeuN, and astrocyte marker glial fibrillary acidic protein in human neural stem cells-treated rats, but not in the untreated ischemic animals. The number of 5-bromo-2′-deoxyuridine-positive ⁄ anti-von Willebrand factor-positive proliferating endothelial cells was higher in the ischemic boundary zone of human neural stem cells-treated rats than in controls. Finally, transplantation of human neural stem cells in the brains of rats with focal cerebral ischemia promoted the proliferation of endogenous neural stem cells and their differentiation into mature neural-like cells, and enhanced angiogenesis. This study provides valuable insights into the effect of human neural stem cell transplantation on focal cerebral ischemia, which can be applied to the development of an effective therapy for stroke.</P>

신경줄기세포의 냉동보관법 확립

권광원(Kwang Won Kwon),김미란(Mi Ran Kim),서해영(Haeyoung Suh-Kim),이영돈(Young Don Lee),김성수(Sung Soo Kim) 대한해부학회 2004 Anatomy & Cell Biology Vol.37 No.6

신경줄기세포(neural stem cells)는 신경세포와 신경아교세포를 형성할 수 있는 능력을 가진 세포로서 발생중인 중추신경계뿐만 아니라 성인 뇌조직의 제한된 부위에서도 발견된다. 본 연구에서는 사람 신경줄기세포의 장기간 보존을 위하여 냉동보관 방법을 확립하였다. 또한 냉동보관된 태아 뇌조직으로부터 신경줄기세포를 분리, 배양할 수 있는 가능성을 조사하였다. 우선 신경줄기세포의 표지자로 알려진 nestin의 발현과 함께 green fluorescence protein (GFP)을 발현하는 형질전환 생쥐를 이용하여 신경구의 형태로 증식된 세포가 신경줄기세포임을 GFP 발현을 통해 확인하였다. 태아의 신경줄기세포는 신경구에서 nestin의 발현을 통해 확인하였고, 분화조건에서 신경세포, 별아교세포, 희소돌기아교세포로 분화하는 것으로 미루어 이들이 다분화능을 유지하고 있음을 확인하였다. 신경구를 냉동보관하고 해동시킨 후 다시 증식시켜 얻은 신경구의 분화능력은 냉동 전의 신경구와 차이를 나타내지 않았다. 또 임신 10~15주의 태아의 대뇌조직을 잘게 부수어 신경구와 동일한 방법으로 냉동보관한 후 이를 해동시켜 신경구의 형성을 유도한 경우에도 신경세포와 별아교세포로의 분화능력이 유지됨을 관찰하였다. 이는 조직에 남아있던 신경줄기세포가 생존하여 증식하였기 때문으로 추정된다. 본 연구의 결과는 뇌조직이나 대량 배양된 신경구를 장기간 냉동보관하여 필요한 시기에 해동, 배양하여 신경줄기세포의 이식에 사용할 수 있다는 가능성을 제시하고 있다. Neural stem cells are multipotent stem cells that can differentiate into neurons and glial cells. Neural stem cells are found in not only developing nervous system but some restricted regions in adult brain. Here, we presented an effective method that allows a long-term preservation of neural stem cells without losing multipotency. First, we isolated neural stem cells from the developing forebrain of nestin-EGFP transgenic mice carrying green fluorescence protein (GFP) driven by nestin promoter and enhancer. Primary neurospheres isolated from these mice highly expressed GFP. The expression of GFP in neurospheres was sustained for several passages. In order to investigate the effect of freezing on the stem cell properties, we cryopreserved the primary neurospheres for 2 wks in liquid nitrogen. GFP expression pattern as well as differentiation potential of the secondary neurosphere formed after cryopreservation were not that different from those of the primary neurosphere formed before cryopreservation. When the same cryopreservation method was applied to neural stem cells isolated from human fetal brain (gestation 13~15 wks), the expression of nestin, a stem cell marker, and differentiation patterns were not changed after cryopreservation. We also performed isolation of neural stem cells from long-term cryopreserved human fetal brain tissues. The neurospheres were successfully formed and showed similar differention properties with neurospheres isolated from fresh brain tissue. In addition, we demonstrated multipotentiality of neural stem cells was not changed with the duration of cryopreservation of brain tissue, suggesting the self renewality and multipotentiality of neural stem cells were not affected by long-term cryopreservation, The present results provide an useful information for the development of stem cell expansion which is essential factor in clinical application of stem cells.

Expression profile of an operationally-defined neural stem cell clone

Parker, Mark A.,Anderson, Julia K.,Corliss, Deborah A.,Abraria, Victoria E.,Sidman, Richard L.,Park, Kook In,Teng, Yang D.,Cotanche, Douglas A.,Snyder, Evan Y. Elsevier 2005 Experimental neurology Vol.194 No.2

<P><B>Abstract</B></P><P>Neural stem cells (NSCs) are the most primordial and least committed cells of the nervous system, the cells that exist <I>before</I> regional specification develops. Because immunocytochemically-detectable markers that are sufficiently specific and sensitive to define an NSC have not yet been fully defined, we have taken the strong view that, to be termed a “stem cell” in the nervous system—in contrast to a “progenitor” or “precursor” (whose lineage commitment is further restricted)—a <I>single neuroectodermally-derived cell</I> must fulfill an operational definition that is essentially similar to that used in hematopoiesis. In other words, it must possess the following functional properties: (1) “Multipotency”, i.e., the ability to yield mature cells in all three fundamental neural lineages throughout the nervous system—neurons (of all subtypes), astrocytes (of all types), oligodendrocytes—in multiple regional and developmental contexts and in a region and developmental stage-appropriate manner. (2) The ability to populate a developing region and/or repopulate an ablated or degenerated region of the nervous system with appropriate cell types. (3) The ability to be serially transplanted. (4) “Self-renewal”, i.e., the ability to produce daughter cells (including new NSCs) with identical properties and potential. Having identified a murine neural cell clone that fulfills this strict operational definition—in contrast to other studies that used less rigorous or non-operational criteria for defining an NSC (e.g., the “neurosphere” assay)—we then examined, by comparing gene expression profiles, the relationship such a cell might have to (a) a <I>multipotent</I> somatic stem cell from another organ system (the hematopoietic stem cell [HSC]); (b) a <I>pluripotent</I> stem cell derived from the inner cell mass and hence without organ assignment (an embryonic stem cell); (c) neural cells isolated and maintained primarily as neurospheres but without having been subjected to the abovementioned operational screen (“CNS-derived neurospheres”). ESCs, HSCs, and operationally-defined NSCs—all of which have been identified not only by markers but by functional assays in their respective systems and whose state of differentiation could be synchronized—shared a large number of genes. Although, as expected, the most stem-like genes were expressed by ESCs, NSCs and HSCs shared a number of genes. CNS-derived neurospheres, on the other hand, expressed fewer “stem-like” genes held in common by the other operationally-defined stem cell populations. Rather they displayed a profile more consistent with differentiated neural cells. (Genes of neural identity were shared with the NSC clone.) Interestingly, when the operationally-defined NSC clone was cultured as a neurosphere (rather than in monolayer), its expression pattern shifted from a “stem-like” pattern towards a more “differentiated” one, suggesting that the neurosphere, without functional validation, may be a poor model for predicting stem cell attributes because it consists of heterogeneous populations of cells, only a small proportion of which are truly “stem-like”. Furthermore, when operational definitions are employed, a common set of stem-like genes does emerge across both embryonic and somatic stem cells of various organ systems, including the nervous system.</P>

Efficient In Vitro Labeling Rabbit Bone Marrow-Derived Mesenchymal Stem Cells with SPIO and Differentiating into Neural-Like Cells

Zhang, Ruiping,Li, Jing,Li, Jianding,Xie, Jun Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.9

Mesenchymal stem cells (MSCs) can differentiate into neural cells to treat nervous system diseases. Magnetic resonance is an ideal means for cell tracking through labeling cells with superparamagnetic iron oxide (SPIO). However, no studies have described the neural differentiation ability of SPIO-labeled MSCs, which is the foundation for cell therapy and cell tracking in vivo. Our results showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) labeled in vitro with SPIO can be induced into neural-like cells without affecting the viability and labeling efficiency. The cellular uptake of SPIO was maintained after labeled BM-MSCs differentiated into neural-like cells, which were the basis for transplanted cells that can be dynamically and non-invasively tracked in vivo by MRI. Moreover, the SPIO-labeled induced neural-like cells showed neural cell morphology and expressed related markers such as NSE, MAP-2. Furthermore, whole-cell patch clamp recording demonstrated that these neural-like cells exhibited electrophysiological properties of neurons. More importantly, there was no significant difference in the cellular viability and $[Ca^{2+}]_i$ between the induced labeled and unlabeled neural-like cells. In this study, we show for the first time that SPIO-labeled MSCs retained their differentiation capacity and could differentiate into neural-like cells with high cell viability and a good cellular state in vitro.

Differentiation Potential of Epiblast Stem Cells into Neural Stem Cells

Hyo Jin Jang,Sol Choi,Jong Soo Kim,Hyun Woo Choi,Jin Young Joo,Min Jung Kim,Jeong Tae Do 한국동물번식학회 2012 Reproductive & Developmental Biology(Supplement) Vol.36 No.2s

Pluripotent stem cells can be derived from both pre- and post-implantation embryos. Embryonic stem cells (ES cells), derived from inner cell mass (ICM) of blastocyst are naïve pluripotent and epiblast stem cells (EpiSCs) derived from post-implantation epiblast are primed pluripotent. The phenotypes and gene expression patterns of the two pluripotent stem cells are different each other and EpiSCs thought to be in a more advanced pluripotent (primed pluripotent state) than mouse ES cells (naïve pluripotent state). Therefore, we questioned whether EpiSCs are less potential to be differentiated into specialized cell types in vitro. EpiSCs were isolated from 5.5~6.5 day post coitum mouse embryos of the post-implantation epiblast. The EpiSCs could differentiate into all tree germ layers in vivo, and expressed pluripotency markers (Oct4, Nanog). Interestingly, EpiSCs also were able to efficiently differentiate into neural stem cells (NSCs). The NSCs differentiated from EpiSCs (EpiSC-NSCs) expressed NSC markers (Nestin, Sox2, and Musasi), self-renewed over passage 20, and could differentiate into two neural subtypes, neurons, astrocytes and oligodendrocytes. Next, we compared global gene expression patterns of EpiSC-NSCs with that of NSCs differentiated from ES cells and brain tissue. Gene expression pattern of brain tissue derived NSCs were closer to ES cell-derived NSCs than EpiSC-NSCs, indicating that the pluripotent stem cell-derived somatic cells could have different characteristics depending on the origin of pluripotent stem cell types. * This work was supported by the Next Generation Bio-Green 21 Program funded by the Rural Development Administration (Grant PJ 008009).

Efficient In Vitro Labeling Rabbit Bone Marrow-Derived Mesenchymal Stem Cells with SPIO and Differentiating into Neural-Like Cells

Ruiping Zhang,Jing Li,Jianding Li,Jun Xie 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.9

Mesenchymal stem cells (MSCs) can differentiate into neural cells to treat nervous system diseases. Magnetic resonance is an ideal means for cell tracking through labeling cells with superparamagnetic iron oxide (SPIO). However, no studies have described the neural differentiation ability of SPIO-labeled MSCs, which is the foundation for cell therapy and cell tracking in vivo. Our results showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) labeled in vitro with SPIO can be induced into neural-like cells without affecting the viability and labeling efficiency. The cellular uptake of SPIO was maintained after labeled BM-MSCs differentiated into neural-like cells, which were the basis for transplanted cells that can be dynamically and non-invasively tracked in vivo by MRI. Moreover, the SPIO-labeled induced neural-like cells showed neural cell morphology and expressed related markers such as NSE, MAP-2. Furthermore, whole-cell patch clamp recording demonstrated that these neural-like cells exhibited electrophysiological properties of neurons. More importantly, there was no significant difference in the cellular viability and [Ca2+]i between the induced labeled and unlabeled neural-like cells. In this study, we show for the first time that SPIO-labeled MSCs retained their differentiation capacity and could differentiate into neural-like cells with high cell viability and a good cellular state in vitro.

Current Status and Future Strategies to Treat Spinal Cord Injury with Adult Stem Cells

Jeong, Seong Kyun,Choi, Il,Jeon, Sang Ryong The Korean Neurosurgical Society 2020 Journal of Korean neurosurgical society Vol.63 No.2

Spinal cord injury (SCI) is one of the most devastating conditions and many SCI patients suffer neurological sequelae. Stem cell therapies are expected to be beneficial for many patients with central nervous system injuries, including SCI. Adult stem cells (ASCs) are not associated with the risks which embryonic stem cells have such as malignant transformation, or ethical problems, and can be obtained relatively easily. Consequently, many researchers are currently studying the effects of ASCs in clinical trials. The environment of transplanted cells applied in the injured spinal cord differs between the phases of SCI; therefore, many researchers have investigated these phases to determine the optimal time window for stem cell therapy in animals. In addition, the results of clinical trials should be evaluated according to the phase in which stem cells are transplanted. In general, the subacute phase is considered to be optimal for stem cell transplantation. Among various candidates of transplantable ASCs, mesenchymal stem cells (MSCs) are most widely studied due to their clinical safety. MSCs are also less immunogenic than neural stem/progenitor cells and consequently immunosuppressants are rarely required. Attempts have been made to enhance the effects of stem cells using scaffolds, trophic factors, cytokines, and other drugs in animal and/or human clinical studies. Over the past decade, several clinical trials have suggested that transplantation of MSCs into the injured spinal cord elicits therapeutic effects on SCI and is safe; however, the clinical effects are limited at present. Therefore, new therapeutic agents, such as genetically enhanced stem cells which effectively secrete neurotrophic factors or cytokines, must be developed based on the safety of pure MSCs.

SIRT1 is required for oncogenic transformation of neural stem cells and for the survival of “cancer cells with neural stemness” in a p53-dependent manner

Lee, Ji-Seon,Park, Jeong-Rak,Kwon, Ok-Seon,Lee, Tae-Hee,Nakano, Ichiro,Miyoshi, Hiroyuki,Chun, Kwang-Hoon,Park, Myung-Jin,Lee, Hong Jun,Kim, Seung U.,Cha, Hyuk-Jin Oxford University Press 2015 Neuro-oncology Vol.17 No.1

<P><B>Background</B></P><P>Cancer stemness, observed in several types of glioma stem cells (GSCs), has been demonstrated to be an important barrier for efficient cancer therapy. We have previously reported that cancerous neural stem cells (F3.Ras.CNSCs), derived from immortalized human neural stem cells by a single oncogenic stimulation, form glial tumors in vivo.</P><P><B>Method</B></P><P>We searched for a commonly expressed stress modulator in both F3.Ras.CNSCs and GSCs and identified silent mating type information regulation 2, homolog (SIRT1) as a key factor in maintaining cancer stemness.</P><P><B>Result</B></P><P>We demonstrate that the expression of SIRT1, expressed in “cancer cells with neural stemness,” is critical not only for the maintenance of stem cells, but also for oncogenic transformation. Interestingly, SIRT1 is essential for the survival and tumorigenicity of F3.Ras.CNSCs and GSCs but not for the U87 glioma cell line.</P><P><B>Conclusion</B></P><P>These results indicate that expression of SIRT1 in cancer cells with neural stemness plays an important role in suppressing p53-dependent tumor surveillance, the abrogation of which may be responsible not only for inducing oncogenic transformation but also for retaining the neural cancer stemness of the cells, suggesting that SIRT1 may be a putative therapeutic target in GSCs.</P>

Human Embryonic Stem Cell-derived Neuroectodermal Spheres Revealing Neural Precursor Cell Properties

한효원,김장환,강만종,문성주,강용국,구덕본,조이숙 한국발생생물학회 2008 발생과 생식 Vol.12 No.1

만능성 인간 배아줄기세포로부터 확립된 신경줄기세포 또는 신경전구세포는 퇴행성 신경질환 세포치료제로 이용될 수 있는 다양한 종류의 신경세포로 분화 유도될 수 있다. 하지만, 인간 배아줄기세포로부터 신경세포를 생산하기 위한 기술은 아직 많은 장애를 가지고 있다. 인간 배아줄기세포 유래 신경전구세포에서 특징적으로 나타나는 신경관 유사로제트에 대한 이해는 인간 배아줄기세포 신경 분화의 효율을 높이는데 유용한 정보를 제공할 것으로 사료된다. 일반적으로 신경로제트 Neural stem/precursor derived from pluripotent human embryonic stem cells (hESCs) has considerable therapeutic potential due to their ability to generate various neural cells which can be used in cell-replacement therapies for neurodegenerative diseases. However, production of neural cells from hESCs remains technically very difficult. Understanding neural-tube like rosette characteristic neural precursor cells from hESCs may provide useful information to increase the efficiency of hESC neural differentiation. Generally, neural rosettes were derived from differentiating hEBs in attached culture system, however this is time-consuming and complicated. Here, we examined if neural rosettes could be formed in suspension culture system by bypassing attachment requirement. First, we tested whether the size of hESC clumps affected the formation of human embryonic bodies (hEBs) and neural differentiation. We confirmed that hEBs derived from square sized hESC clumps were effectively differentiated into neural lineage than those of the other sizes. To induce the rosette formation, regular size hEBs were derived by incubation of hESC clumps() in EB medium for 1 wk in a suspended condition on low attachment culture dish and further incubated for additional wks in neuroectodermal sphere(NES)-culture medium. We observed the neural tube-like rosette structure from hEBs after days of differentiation. Their identity as a neural precursor cells was assessed by measuring their expressions of neural precursor markers(Vimentin, Nestin, MSI1, MSI2, Prominin-1, Pax6, Sox1, N-cadherin, Otx2, and Tuj1) by RT-PCR and immunofluorescence staining. We also confirmed that neural rosettes could be terminally differentiated into mature neural cell types by additional incubation for wks with NES medium without growth factors. Neuronal(Tuj1, MAP2, GABA) and glial( and GFAP) markers were highly expressed after and 4 wks of incubation, respectively. Expression of oligodendrocyte markers O1 and CNPase was significantly increased after wks of incubation. Our results demonstrate that rosette forming neural precursor cells could be successfully derived from suspension culture system and that will not only help us understand the neural differentiation process of hESCs but also simplify the derivation process of neural precursors from hESCs.

In Vitro Expansion of Homogeneous Neural Precursor Cells Derived from Human Embryonic Stem Cells

Deuk-Chae Na,Sehee Kim,Won-Ik Choi,Hyun-Jin Hwang,Inho Han,Jae-hwan Kim,Keun-Hong Park,Hyung-Min Chung,Seong-Jun Choi 한국동물생명공학회(구 한국동물번식학회) 2007 Reproductive & developmental biology Vol.31 No.4

Human embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and have the capacity to differentiate into various types of cells in the body. Hence, these cells may potentially be an indefinite source of cells for cell therapy in various degenerative diseases including neuronal disorders. For clinical applications of human ES cells, directed differentiation of these cells would be necessary. The objective of this study is to develop the culture condition for the expansion of neural precursor cells derived from human ES cells. Human ES cells were able to differentiate into neural precursor cells upon a stepwise culture condition. Neural precursor cells were propagated up to 5000-fold in cell numbers over 12-week period of culture and evaluated for their characteristics. Expressions of sox1 and pax6 transcripts were dramatically up-regulated along the differentiation stages by RT-PCR analysis. In contrast, expressions of oct4 and nanog transcripts were completely disappeared in neural precursor cells. Expressions of nestin, pax6 and sox1 were also confirmed in neural precursor cells by immunocytochemical analysis. Upon differentiation, the expanded neural precursor cells differentiated into neurons, astrocytes, and oligodendrocytes. In immunocytochemical analysis, expressions of type III β-tubulin and MAP2ab were observed. Presence of astrocytes and oligodendrocytes were also confirmed by expressions of GFAP and O4, respectively. Results of this study demonstrate the feasibility of long-term expansion of human ES cell-derived neural precursor cells in vitro, which can be a potential source of the cells for the treatment of neurodegenerative disorders.