Expansion of retinal stem cells and their progeny using cell microcarriers in a bioreactor

Blindness as a consequence of degenerative eye diseases (e.g., age-related macular degeneration and retinitis pigmentosa) is a major health problem and numbers are expected to increase by up to 50% by 2020. Unfortunately, adult mouse and human retinal stem cells (RSCs), unlike fish and amphibians , are quiescent in vivo and do not regenerate following disease or injury. To replace lost cells, we used microcarriers (MCs) in a suspension stirring bioreactor to help achieve numbers suitable for differentiation and transplantation. We achieved a significant 10-fold enrichment of RSC yield compared to conventional static culture techniques using a combination of FACTIII MCs and relative hypoxia (5%) inside the bioreactor. We found that hypoxia (5% O₂) was associated with better RSC expansion across all platforms; and this can be attributed to hypoxia-induced increases in survival and/or symmetric division of stem cells. In the future, we will target the differentiation of RSCs and their progeny toward rod and cone photoreceptor phenotypes using FACTIII MCs inside bioreactors to expand their populations in order to produce the large numbers of cells needed for transplantation.     

Bone marrow mesenchymal stem cells modified by angiogenin-1 promotes tissue repair in mice with oxygen-induced retinopathy of prematurity by promoting retinal stem cell proliferation and differentiation

Retinopathy has become one of the major factors that lead to blindness worldwide. Although many clinical therapies are concerned about such disease, most of them focus on symptoms alleviation. In this study, we aim to investigate whether coculture retinal stem cells (RSCs) with bone marrow mesenchymal stem cells transfected with angiogenin-1 (Ang-1-BMSCs) affects the damaged retinal tissue of oxygen-induced retinopathy of prematurity (OIR-ROP) mice. After OIR-ROP mouse model establishment, Ang-1-BMSCs, RSCs, and OIR-ROP retinal tissues were cocultured in a a transwell chamber. RSCs proliferation and the expression of Ang-1, insulin-like growth factor-1 (IGF-1) in the supernatant of RSCs, as well as β-tubulin and protein kinase C (PKC) expression were evaluated. Finally, the repair of OIR-ROP mice retinal tissues was observed by injecting Ang-1-BMSCs + RSCs. In the OIR-ROP mouse model, RSCs cocultured with OIR-ROP retinal tissues could be induced to differentiate into cells expressing β-tubulin and PKC and promote the expression of Ang-1 and IGF-1. coculture of Ang-1-BMSCs further enhanced the proliferation and differentiation of RSCs by promoting the expression of Ang-1 and IGF-1. Coculture of RSCs + Ang-1-BMSCs induced differentiation of Ang-1-BMSCs through interaction among intercellular factors and restored the damaged retinal tissue of OIR-ROP mice. Collectively, our study provided evidence that coculture of Ang-1-BMSCs and RSCs could promote the proliferation and differentiation of RSCs and improve the treatment for the damaged retina tissue of OIR-ROP mice.     

Otx2 enhances transdifferentiation of Müller cells‐derived retinal stem cells into photoreceptor‐like cells

Retinal Müller glial cells have the potential of neurogenic retinal progenitor cells, and could reprogram into retinal‐specific cell types such as photoreceptor cells. How to promote the differentiation of Müller cells into photoreceptor cells represents a promising therapy strategy for retinal degeneration diseases. This study aimed to enhance the transdifferentiation of rat Müller cells‐derived retinal stem cells (MC‐RSCs) into photoreceptor‐like cells and explore the signalling mechanism. We dedifferentiated rat Müller cells into MC‐RSCs which were infected with Otx2 overexpression lentivirus or control. The positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus was significantly higher compared to control. Furthermore, pre‐treatment with Crx siRNA, Nrl siRNA, or GSK‐3 inhibitor SB‐216763 reduced the positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus. Finally, Otx2 induced photoreceptor precursor cells were injected into subretinal space of N‐methyl‐N‐nitrosourea induced rat model of retinal degeneration and partially recovered retinal degeneration in the rats. In conclusion, Otx2 enhances transdifferentiation of MC‐RSCs into photoreceptor‐like cells and this is associated with the inhibition of Wnt signalling. Otx2 is a potential target for gene therapy of retinal degenerative diseases.     

人胚与鼠胚神经干细胞体外培养的差异

为比较人胚与鼠胚神经干细胞体外培养的差异。实验采用具有丝裂原作用的细胞生长因子。结合无血清细胞培养技术从人胚和鼠胚皮层分离神经干细胞。在连续传代过程中观察其体外培养特性,免疫荧光染色检测Nestin抗原和分化后特异性成熟神经细胞抗原的表达,并用流式细胞仪检测神经干细胞分化情况。结果表明：（1）使用单一生长因子即可从鼠胚皮层分离神经干细胞,但在人胚却需同时使用多种生长因子,协同使用bFGF,EGF和LIF是人胚神经干细胞体外培养的较佳条件;（2）鼠胚皮层神经干细胞在连续传代过程中增殖速度快于人胚,其Nestin阳性率和BrdU标记的阳性率亦高于人胚,表明其增殖能力明显高于人胚,（3）人胚神经干细胞较鼠胚更易分化为神经元。     

多能成体祖细胞培养条件促进人脂肪干细胞的视网膜神经保护功能

目的：检测多能成体祖细胞（MAPC）的培养条件对猴骨髓间充质细胞（BMMSCs）和人脂肪干细胞（hADSCs）生长的影响,旨在获得更适合治疗视网膜变性疾病的供体细胞。方法通过细胞形态观察、MTT实验、克隆形成率、PCR检测、以及成脂、成骨、成软骨分化潜能检测等,研究MAPC培养条件下猴BMMSCs和hADSCs的特征,并用DMEM/LG和MAPC培养条件培养的hADSCs进行RCS大鼠视网膜下腔移植,通过视网膜电图（ERG）和TUNEL检测,判断细胞移植治疗对视功能及视网膜细胞凋亡的影响。结果与常规培养基相比,MAPC培养条件能促进猴BMMSCs增殖,细胞变小,但传2代后,细胞变得宽大扁平,出现衰老征象;然而,MAPC培养条件下的hADSCs细胞增殖能力及克隆形成率均增强,形成的克隆较大可稳定传10代以上,且具有成脂、成骨、成软骨的多向分化潜能,细胞表面标记物及细胞因子出现差异表达：CD140b、CD90、CD47、HGF和PEDF显著上调,CD73、CD105和IL-6显著下调。与对照组相比,移植DMEM/LG和MAPC培养条件培养的hADSCs（P4）3周后,RCS大鼠的B波波幅明显升高,外核层细胞凋亡明显减少。结论 MAPC培养条件培养的hADSCs显示出更好的视网膜神经保护作用,适合用于治疗视网膜退行性疾病。     

Retinal stem cells: promising candidates for retina transplantation

Stem cell transplantation is widely considered as a promising therapeutic approach for photoreceptor degeneration, one of the major causes of blindness. In this review, we focus on the biology of retinal stem cells (RSCs) and progenitor cells (RPCs) isolated from fetal, postnatal, and adult animals, with emphasis on those from rodents and humans. We discuss the origin of RSCs/RPCs, the markers expressed by these cells and the conditions for the isolation, culture, and differentiation of these cells in vitro or in vivo by induction with exogenous stimulation. Commercial disclosure: none.     

Step-wise specification of retinal stem cells during normal embryogenesis

The specification of embryonic cells to produce the retina begins at early embryonic stages as a multi-step process that gradually restricts fate potentials. First, a subset of embryonic cells becomes competent to form retina by their lack of expression of endo-mesoderm-specifying genes. From these cells, a more restricted subset is biased to form retina by virtue of their close proximity to sources of bone morphogenetic protein antagonists during neural induction. During gastrulation, the definitive RSCs (retinal stem cells) are specified as the eye field by interactions with underlying mesoderm and the expression of a network of retina-specifying genes. As the eye field is transformed into the optic vesicle and optic cup, a heterogeneous population of RPCs (retinal progenitor cells) forms to give rise to the different domains of the retina: the optic stalk, retinal pigmented epithelium and neural retina. Further diversity of RPCs appears to occur under the influences of cell-cell interactions, cytokines and combinations of regulatory genes, leading to the differentiation of a multitude of different retinal cell types. This review examines what is known about each sequential step in retinal specification during normal vertebrate development, and how that knowledge will be important to understand how RSCs might be manipulated for regenerative therapies to treat retinal diseases.     

Fate restriction and multipotency in retinal stem cells

Stem cells have the capacity to both self-renew and generate postmitotic cells. Long-term tracking of individual clones in their natural environment constitutes the ultimate way to validate postembryonic stem cells. We identify retinal stem cells (RSCs) using the spatiotemporal organization of the fish retina and follow the complete offspring of a single cell during the postnatal life. RSCs generate two tissues of the adult fish retina, the neural retina (NR) and the retinal-pigmented epithelium (RPE). Despite their common embryonic origin and tight coordination during continuous organ growth, we prove that NR and RPE are maintained by dedicated RSCs that contribute in a fate-restricted manner to either one or the other tissue. We show that in the NR, RSCs are multipotent and generate all neuron types and glia. The clonal origin of these different cell types from a multipotent NSC has far-reaching implications for cell type and tissue homeostasis.     

Loss of FGF-Dependent Mesoderm Identity and Rise of Endogenous Retinoid Signalling Determine Cessation of Body Axis Elongation

The endogenous mechanism that determines vertebrate body length is unknown but must involve loss of chordo-neural-hinge (CNH)/axial stem cells and mesoderm progenitors in the tailbud. In early embryos, Fibroblast growth factor (FGF) maintains a cell pool that progressively generates the body and differentiation onset is driven by retinoid repression of FGF signalling. This raises the possibility that FGF maintains key tailbud cell populations and that rising retinoid activity underlies cessation of body axis elongation. Here we show that sudden loss of the mesodermal gene (Brachyury) from CNH and the mesoderm progenitor domain correlates with FGF signalling decline in the late chick tailbud. This is accompanied by expansion of neural gene expression and a similar change in cell fate markers is apparent in the human tailbud. Fate mapping of chick tailbud further revealed that spread of neural gene expression results from continued ingression of CNH-derived cells into the position of the mesoderm progenitor domain. Using gain and loss of function approaches in vitro and in vivo, we then show that attenuation of FGF/Erk signalling mediates this loss of Brachyury upstream of Wnt signalling, while high-level FGF maintains Brachyury and can induce ectopic CNH-like cell foci. We further demonstrate a rise in endogenous retinoid signalling in the tailbud and show that here FGF no longer opposes retinoid synthesis and activity. Furthermore, reduction of retinoid signalling at late stages elevated FGF activity and ectopically maintained mesodermal gene expression, implicating endogenous retinoid signalling in loss of mesoderm identity. Finally, axis termination is concluded by local cell death, which is reduced by blocking retinoid signalling, but involves an FGFR-independent mechanism. We propose that cessation of body elongation involves loss of FGF-dependent mesoderm identity in late stage tailbud and provide evidence that rising endogenous retinoid activity mediates this step and ultimately promotes cell death in chick tailbud.     

Generation of mesenchymal stem cell lines from murine bone marrow

Mesenchymal stem cells (MSC), because of their multipotency and ease of purification and amplification, are an ideal stem cell source for cell therapies. Bone-marrow-derived stem cells (BMSC) can be used to develop MSC-like immortalized cell lines with large proliferation and differentiation potentialities. Their immortalized status prevents the maintenance of MSC function and characters; this can be negated by modifying the isolation and maintenance protocol. Adult murine BMSC were isolated and maintained in media without additional growth factors together with passage-dependent reseeding following trypsinization. Cells maintained over 25 passages were considered as putative cell lines and characterized. The phenotypic and genotypic characteristics and multilineage differentiation potential of the cells were assessed by morphological, phenotypic, and molecular assays at various passages. The putative BMSC cell lines showed the characteristics of MSC and were able to maintain these characteristics, even after immortalization. The phenotypic data demonstrated difference among two cell lines; this was further validated by the difference in their multilineage differentiation potential following specific induction. More importantly, no changes were observed in the genotypic level in comparison with control cells, even after more than 50 passages. Our protocol thus advances the isolation and maintenance of BMSC and the development of putative BMSC cell lines that maintain characteristics of MSC, including multilineage differentiation potential, after more than 40 passages.     

表皮生长因子诱导骨髓间充质干细胞向视网膜神经细胞分化的体外研究

目的:研究表皮生长因子诱导骨髓间充质干细胞向视网膜神经细胞分化的可能性。方法:体外培养骨髓间充质干细胞,利用流式细胞仪分析其细胞表型。采用含EGF的培养液诱导骨髓间充质干细胞向视网膜神经细胞分化,并利用免疫荧光法进行鉴定。结果:从骨髓中分离培养的细胞具有成纤维细胞样形态,贴壁生长,表型相对均一,表面标志为CD90、CD44、CD147阳性;而CD34、CD38、CD45、CD14、HLA-DR阴性。体外诱导后可以得到神经干细胞标志物nestin、神经胶质细胞标志物GFAP和视网膜光感受器细胞标志物Rhodopsin呈阳性表达的细胞。结论:从骨髓中分离培养得到的间充质干细胞具有向视网膜神经细胞分化的潜能。     

Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons

Human embryonic stem (hES) cells can be guided to differentiate into ventral midbrain-type neural precursor (NP) cells that proliferate in vitro by specific mitogens. We investigated the potential of these NP cells derived from hES cells (hES-NP) for the large-scale generation of human dopamine (DA) neurons for functional analyses and therapeutic applications. To address this, hES-NP cells were expanded in vitro for 1.5 months with six passages, and their proliferation and differentiation properties determined over the NP passages. Interestingly, the total hES-NP cell number was increased by > 2 × 10⁴-folds over the in vitro period without alteration of phenotypic gene expression. They also sustained their differentiation capacity toward neuronal cells, exhibiting in vitro pre-synaptic DA neuronal functionality. Furthermore, the hES-NP cells can be cryopreserved without losing their proliferative and developmental potential. Upon transplantation into a Parkinson's disease rat model, the multi-passaged hES-NP cells survived, integrated into the host striatum, and differentiated toward the neuronal cells expressing DA phenotypes. A significant reduction in the amphetamine-induced rotation score of Parkinson's disease rats was observed by the cell transplantation. Taken together, these findings indicate that hES-NP cell expansion is exploitable for a large-scale generation of experimental and transplantable DA neurons of human-origin.     

c-kit marks late retinal progenitor cells and regulates their differentiation in developing mouse retina

Retinal progenitor cells are believed to display altered proliferation and differentiation during retinal development, suggesting that retinal progenitor cell populations are not homogeneous. However, the composition of progenitor cell populations is not known, due in part to the lack of known surface markers identifying distinct stages of retinal progenitor cells. We found a dramatic change in the expression profile of the cell surface antigens c-kit and stage-specific embryonic antigen-1 (SSEA-1) in retinal progenitor cells during development. While SSEA-1 was expressed early in development, c-kit expression peaked in late stage progenitor cells. The identification of these developmental markers enabled us to characterize distinct sub-populations of retinal progenitor cells. Progenitor cell subpopulations expressing either SSEA-1, c-kit, or both showed different proliferation and differentiation abilities. Although SSEA-1-positive cells were augmented by beta-catenin signaling, c-kit-positive cells were positively regulated by Notch signaling. Taken together, our data suggest that c-kit and SSEA-1 can be used to spatiotemporally differentiate retinal progenitor populations that have intrinsically distinct characteristics. Prolonged expression of c-kit by a retrovirus resulted in the promotion of proliferation and the appearance of nestin-positive cells in the presence of the c-kit ligand, stem cell factor (SCF). This suggests a role for c-kit, Notch, and the beta-catenin signaling network in retinal development.     

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.     

Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles

We previously demonstrated that keratin 15 expressing cells present in the bulge region of hair follicles exhibit properties of adult stem cells. We have now established and characterized an immortalized adult epithelial stem cell line derived from cells isolated from the human hair follicle bulge region. Telogen hair follicles from human skin were microdissected to obtain an enriched population of keratin 15 positive skin stem cells. By expressing human papillomavirus 16 E6/E7 genes in these stem cells, we have been able to culture the cells for >30 passages and maintain a stable phenotype after 12 mo of continuous passage. The cell line was compared to primary stem cells for expression of stem cell specific proteins, for in vitro stem cell properties, and for their capacity to differentiate into different cell lineages. This new cell line, named Tel-E6E7 showed similar expression patterns to normal skin stem cells and maintained in vitro properties of stem cells. The cells can differentiate into epidermal, sebaceous gland, and hair follicle lineages. Intact beta-catenin dependent signaling, which is known to control in vivo hair differentiation in rodents, is maintained in this cell line. The Tel-E6E7 cell line may provide the basis for valid, reproducible in vitro models for studies on stem cell lineage determination and differentiation.     

In vitro differentiation of retinal pigment epithelium from adult retinal stem cells

One of the limitations in molecular and functional studies of the retinal pigment epithelium (RPE) has been the lack of an in vitro system retaining all the features of in vivo RPE cells. Retinal pigment epithelium cell lines do not show characteristics typical of a functional RPE, such as pigmentation and expression of specific markers. The present study was aimed at the development of culture conditions to differentiate, in vitro, retinal stem cells (RSC), derived from the adult ciliary body, into a functional RPE. Retinal stem cells were purified from murine eyes, grown as pigmented neurospheres and induced to differentiate into RPE on an extracellular matrix substrate using specific culture conditions. After 7-15 days of culture, pigmented cells with an epithelial morphology showed a polarized organization and a capacity for phagocytosis. We detected different stages of melanogenesis in cells at 7 days of differentiation, whereas RPE at 15 days contained only mature melanosomes. These data suggest that our protocol to differentiate RPE in vitro can provide a useful model for molecular and functional studies.     

Effects of membrane reference state on shape memory of a red blood cell

By using a three-dimensional continuum model, we simulate the shape memory of a red blood cell after the remove of external forces. The purpose of this study is to illustrate the effect of membrane reference state on cell behavior during the recovery process. The reference state of an elastic element is the geometry with zero stress. Since the cell membrane is composed of cytoskeleton and lipid bilayer, both the reference states of cytoskeleton (RSC) and lipid bilayer (RSL) are considered. Results show that a non-spherical RSC can result in shape memory. The energy barrier due to non-spherical RSC is determined by the ratio of the equator length to the meridian length of the RSC. Thus different RSCs can have similar energy barrier and leading to identical recovery response. A series of simulations of more intermediate RSCs show that the recovery time scale is inversely proportional to the energy barrier. Comparing to spherical RSL, a spheroid RSL contributes to the energy barrier and recovery time. Furthermore, we observe a folding recovery due to the biconcave RSL which is different from the tank treading recovery. These results may motivate novel numerical and experimental studies to determine the exact RSC and RSL.     

小分子化合物诱导干细胞向视网膜感光细胞分化的研究进展

干细胞是一类具有多向分化潜能的细胞群,如胚胎干细胞(embryonic stem cell,ESC)、诱导多潜能干细胞(induced pluripotent stem cell,i PSC)等,可在特定的条件下向包括视网膜感光细胞在内的多种细胞分化。小分子化合物是一类由组织细胞合成、分泌的小分子多肽类因子,特定的小分子化合物可作用于干细胞诱导其向视网膜感光细胞分化。目前,对干细胞体外培养,通过使用不同的诱导培养方案,探索干细胞向视网膜感光细胞分化的研究成为热点。早期,研究者们主要在共培养条件下采用小分子化合物诱导ESC向视网膜感光细胞分化,随着研究的进展,逐渐开始探索在无共培养条件下小分子化合物诱导ESC向视网膜感光细胞的分化以及小分子化合物诱导i PSC向视网膜感光细胞的分化。本文主要就小分子化合物促进ESC和i PSC向视网膜感光细胞分化的研究进展进行综述。