A new efficient protocol for directed differentiation of retinal pigmented epithelial cells from normal and retinal disease induced pluripotent stem cells

We describe a new, efficient protocol that involves the serial addition of noggin, basic fibroblast growth factor (bFGF), retinoic acid, and sonic hedgehog (Shh) for the differentiation of human induced pluripotent stem cells (hiPSC) to retinal pigmented epithelium (RPE) in a serum- and feeder-free adherent condition. hiPSC-RPE cells exhibited RPE morphology and specific molecular markers. Additionally, several hiPSC lines were generated from retinal-specific patients with Leber's congenital amaurosis, Usher syndrome, two patients with retinitis pigmentosa, and a patient with Leber's hereditary optic neuropathy. The RPE cells generated from these disease-specific hiPSCs expressed specific markers by the same RPE lineage-directed differentiation protocol. These findings indicate a new short-term, simple, and efficient protocol for differentiation of hiPSCs to RPE cells. Such specific retinal disease-specific hiPSCs offer an unprecedented opportunity to recapitulate normal and pathologic formation of human retinal cells in vitro, thereby enabling pharmaceutical screening, and potentially autologous cell replacement therapies for retinal diseases.     

Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment

Differentiation methods for human induced pluripotent stem cells (hiPSCs) typically yield progeny from multiple tissue lineages, limiting their use for drug testing and autologous cell transplantation. In particular, early retina and forebrain derivatives often intermingle in pluripotent stem cell cultures, owing to their shared ancestry and tightly coupled development. Here, we demonstrate that three-dimensional populations of retinal progenitor cells (RPCs) can be isolated from early forebrain populations in both human embryonic stem cell and hiPSC cultures, providing a valuable tool for developmental, functional, and translational studies. Using our established protocol, we identified a transient population of optic vesicle (OV)-like structures that arose during a time period appropriate for normal human retinogenesis. These structures were independently cultured and analyzed to confirm their multipotent RPC status and capacity to produce physiologically responsive retinal cell types, including photoreceptors and retinal pigment epithelium (RPE). We then applied this method to hiPSCs derived from a patient with gyrate atrophy, a retinal degenerative disease affecting the RPE. RPE generated from these hiPSCs exhibited a disease-specific functional defect that could be corrected either by pharmacological means or following targeted gene repair. The production of OV-like populations from human pluripotent stem cells should facilitate the study of human retinal development and disease and advance the use of hiPSCs in personalized medicine.     

人包皮成纤维细胞来源的多能性干细胞的建立

目的 探讨人包皮成纤维细胞重编程为诱导多能性干细胞(iPSCs)的方法.方法 利用反转录病毒携带绿色荧光蛋白(GFP)作为指示,转导到人包皮成纤维细胞中,确定最佳的病毒感染剂量,应用经典的Yamanaka方法,联合小分子物质组蛋白去乙酰化酶抑制剂丙戊酸盐( VPA)诱导人包皮成纤维细胞为多能性干细胞.通过碱性磷酸酶(AKP)染色及免疫荧光细胞化学方法,检测人胚胎干细胞多能性标记物在所建立的诱导多能性干细胞(iPSCs)中的表达,RT-PCR检测拟胚体(EB)三胚层特异基因的表达.结果 以GFP作为指示,建立了高效的反转录病毒感染体系,将人包皮成纤维细胞重编程为iPSCs;所建立的iPSCsAKP染色阳性,表达人胚胎干细胞多能性标记物OCT4、TRA-1-60和TRA-1-81,体外诱导分化后具有三胚层特异基因的表达;小分子物质VPA的添加并未显著提高重编程效率.结论 在GFP所指示的最佳病毒感染剂量下,联合小分子物质VPA成功建立了人包皮成纤维细胞来源的iPSCs.     

Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium

We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40–60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.     

Differentiation and transplantation of functional pancreatic Beta cells generated from induced pluripotent stem cells derived from a type 1 diabetes mouse model

The nonobese diabetic (NOD) mouse is a classical animal model for autoimmune type 1 diabetes (T1D), closely mimicking features of human T1D. Thus, the NOD mouse presents an opportunity to test the effectiveness of induced pluripotent stem cells (iPSCs) as a therapeutic modality for T1D. Here, we demonstrate a proof of concept for cellular therapy using NOD mouse-derived iPSCs (NOD-iPSCs). We generated iPSCs from NOD mouse embryonic fibroblasts or NOD mouse pancreas-derived epithelial cells (NPEs), and applied directed differentiation protocols to differentiate the NOD-iPSCs toward functional pancreatic beta cells. Finally, we investigated whether the NPE-iPSC-derived insulin-producing cells could normalize hyperglycemia in transplanted diabetic mice. The NOD-iPSCs showed typical embryonic stem cell-like characteristics such as expression of markers for pluripotency, in vitro differentiation, teratoma formation, and generation of chimeric mice. We developed a method for stepwise differentiation of NOD-iPSCs into insulin-producing cells, and found that NPE-iPSCs differentiate more readily into insulin-producing cells. The differentiated NPE-iPSCs expressed diverse pancreatic beta cell markers and released insulin in response to glucose and KCl stimulation. Transplantation of the differentiated NPE-iPSCs into diabetic mice resulted in kidney engraftment. The engrafted cells responded to glucose by secreting insulin, thereby normalizing blood glucose levels. We propose that NOD-iPSCs will provide a useful tool for investigating genetic susceptibility to autoimmune diseases and generating a cellular interaction model of T1D, paving the way for the potential application of patient-derived iPSCs in autologous beta cell transplantation for treating diabetes.     

Induction of pluripotent stem cells from autopsy donor-derived somatic cells

Human induced pluripotent stem cells (iPSCs) have become an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors’ complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. In this study, we describe iPSCs generated from a skin biopsy collected postmortem during the rapid autopsy of a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. These iPSCs were established in a feeder-free system by lentiviral transduction of the Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Selected iPSC clones expressed both nuclear and surface antigens recognized as pluripotency markers of human embryonic stem cells (hESCs) and were able to differentiate in vitro into neurons and glia. Statistical analysis also demonstrated that fibroblast proliferation was significantly affected by biopsy site, but not donor age (within an elderly cohort). These results provide evidence that autopsy donor-derived fibroblasts can be successfully reprogrammed into iPSCs, and may provide an advantageous approach for generating iPSC-based neurological disease models.     

Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina

Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments using stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with induced pluripotent stem cells (iPSCs) of swine. Here, we subjected iPSCs of swine to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real-time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG, and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO, and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of rhodopsin (RHO) and rod outer segment-specific membrane protein 1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that iPSCs of swine can differentiate into photoreceptors in culture, and these cells can integrate into the damaged swine neural retina, thus, laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.     

Development of histocompatible primate-induced pluripotent stem cells for neural transplantation

Immune rejection and risk of tumor formation are perhaps the greatest hurdles in the field of stem cell transplantation. Here, we report the generation of several lines of induced pluripotent stem cells (iPSCs) from cynomolgus macaque (CM) skin fibroblasts carrying specific major histocompatibility complex (MHC) haplotypes. To develop a collection of MHC-matched iPSCs, we genotyped the MHC locus of 25 CMs by microsatellite polymerase chain reaction analysis. Using retroviral infection of dermal skin fibroblasts, we generated several CM-iPSC lines carrying different haplotypes. We characterized the immunological properties of CM-iPSCs and demonstrated that CM-iPSCs can be induced to differentiate in vitro along specific neuronal populations, such as midbrain dopaminergic (DA) neurons. Midbrain-like DA neurons generated from CM-iPSCs integrated into the striatum of a rodent model of Parkinson's disease and promoted behavioral recovery. Importantly, neither tumor formation nor inflammatory reactions were observed in the transplanted animals up to 6 months after transplantation. We believe that the generation and characterization of such histocompatible iPSCs will allow the preclinical validation of safety and efficacy of iPSCs for neurodegenerative diseases and several other human conditions in the field of regenerative medicine.     

Enhanced neurotrophin synthesis and molecular differentiation in non-transformed human retinal progenitor cells cultured in a rotating bioreactor

One approach to the treatment of retinal diseases, such as retinitis pigmentosa, is to replace diseased or degenerating cells with healthy cells. Even if all of the problems associated with tissue transplant were to be resolved, the availability of tissue would remain an ongoing problem. We have previously shown that transformed human retinal cells can be grown in a NASA-developed horizontally rotating culture vessel (bioreactor) to form three-dimensional-like structures with the expression of several retinal specific proteins. In this study, we have investigated growth of non-transformed human retinal progenitors (retinal stem cells) in a rotating bioreactor. This rotating culture vessel promotes cell-cell interaction between similar and dissimilar cells. We cultured retinal progenitors (Ret 1-4) alone or as a co-culture with human retinal pigment epithelial cells (RPE, D407) in this system to determine if 3D structures can be generated from non-transformed progenitors. Our second goal was to determine if the formation of 3D structures correlates with the upregulation of neurotrophins, basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGFalpha), ciliary neurotrophic factor (CNTF), and brain-delivered neurotrophic factor (BDNF). These factors have been implicated in progenitor cell proliferation, commitment, differentiation, and survival. We also investigated the expression of the following retinal specific proteins in this system: neuron specific enolase (NSE); tyrosine hydroxylase (TH); D(2)D(3), D(4) receptors; protein kinase-C alpha (PKCalpha), and calbindin. The 3D structures generated were characterized by phase and scanning transmission electron microscopy. Retinal progenitors, cultured alone or as a co-culture in the rotating bioreactor, formed 3D structures with some degree of differentiation, accompanied by the upregulation of bFGF, CNTF, and TGFalpha. Brain-derived neurotrophic factor, which is expressed in vivo in RPE (D407), was not expressed in monolayer cultures of RPE but expressed in the rotating bioreactor-cultured RPE and retinal progenitors (Ret 1-4). Upregulation of neurotrophins was noted in all rotating bioreactor-cultured cells. Also, upregulation of D(4) receptor, calbindin, and PKCalpha was noted in the rotating bioreactor-cultured cells. We conclude that non-transformed retinal progenitors can be grown in the rotating bioreactor to form 3D structures with some degree of differentiation. We relied on molecular and biochemical analysis to characterize differentiation in cells grown in the rotating bioreactor.     

质粒重编程诱导多潜能干细胞及定向分化神经干细胞

目的 　探讨游离质粒载体重编程诱导小鼠胚胎成纤维细胞(mouse embryonic fibroblasts, MEFs)为非整合型诱导多潜能干细胞,并在体外定向分化为神经干细胞(neural stem cells, NSCs), 为神经干细胞移植治疗神经损伤提供稳定、安全的细胞来源。 方法　使用电转仪将质粒pEP4-EO2S-ET2K转入小鼠MEFs, 经诱导培养重编程为诱导多潜能干细胞(induced pluripotent stem cells, iPSCs), iPSCs在不同诱导培养基中经2次悬浮及贴壁培养分化为NSCs,在体内及体外实验鉴定iPSCs多向分化潜能特性及NSCs特性。 结果　体内外实验显示iPSCs具有与胚胎干细胞(embryonic stem cells, ESCs)相似的多向分化潜能, 且不整合外源性基因。iPSCs进一步分化的NSCs其相关标志基因表达与野生型NSCs相近,且较iPSCs显著增加,免疫荧光显示NSCs高表达NSC标志物 NESTIN及PAX6,在体外存活能分化为神经元、少突胶质细胞及星形胶质细胞。 结论　游离质粒能重编程诱导非整合型iPSCs, 并定向分化为神经干细胞及神经元, 是神经损伤修复的理想种子细胞。     

Human induced pluripotent stem-derived retinal pigment epithelium (RPE) cells exhibit ion transport, membrane potential, polarized vascular endothelial growth factor secretion, and gene expression pattern similar to native RPE

Age-related macular degeneration (AMD) is one of the major causes of blindness in aging population that progresses with death of retinal pigment epithelium (RPE) and photoreceptor degeneration inducing impairment of central vision. Discovery of human induced pluripotent stem (hiPS) cells has opened new avenues for the treatment of degenerative diseases using patient-specific stem cells to generate tissues and cells for autologous cell-based therapy. Recently, RPE cells were generated from hiPS cells. However, there is no evidence that those hiPS-derived RPE possess specific RPE functions that fully distinguish them from other types of cells. Here, we show for the first time that RPE generated from hiPS cells under defined conditions exhibit ion transport, membrane potential, polarized vascular endothelial growth factor secretion, and gene expression profile similar to those of native RPE. The hiPS-RPE could therefore be a very good candidate for RPE replacement therapy in AMD. However, these cells show rapid telomere shortening, DNA chromosomal damage, and increased p21 expression that cause cell growth arrest. This rapid senescence might affect the survival of the transplanted cells in vivo and therefore, only the very early passages should be used for regeneration therapies. Future research needs to focus on the generation of "safe" as well as viable hiPS-derived somatic cells.     

Stem Cell Derived Retinal Pigment Epithelium: The Role of Pigmentation as Maturation Marker and Gene Expression Profile Comparison with Human Endogenous Retinal Pigment Epithelium.

In age-related macular degeneration (AMD) the retinal pigment epithelium (RPE) deteriorates, leading to photoreceptor decay and severe vision loss. New therapeutic strategies aim at RPE replacement by transplantation of pluripotent stem cell (PSC)-derived RPE. Several protocols to generate RPE have been developed where appearance of pigmentation is commonly used as indicator of RPE differentiation and maturation. It is, however, unclear how different pigmentation stages reflect developmental stages and functionality of PSC-derived RPE cells. We generated human embryonic stem cell-derived RPE (hESC-RPE) cells and investigated their gene expression profiles at early pigmentation (EP) and late pigmentation (LP) stages. In addition, we compared the hESC-RPE samples with human endogenous RPE. We used a common reference design microarray (44 K). Our analysis showed that maturing hESC-RPE, upon acquiring pigmentation, expresses markers specific for human RPE. Interestingly, our analysis revealed that EP and LP hESC-RPE do not differ much in gene expression. Our data further showed that pigmented hESC-RPE has a significant lower expression than human endogenous RPE in the visual cycle and oxidative stress pathways. In contrast, we observed a significantly higher expression of pathways related to the process adhesion-to-polarity model that is typical of developing epithelial cells. We conclude that, in vitro, the first appearance of pigmentation hallmarks differentiated RPE. However, further increase in pigmentation does not result in much significant gene expression changes and does not add important RPE functionalities. Consequently, our results suggest that the time span for obtaining differentiated hESC-RPE cells, that are suitable for transplantation, may be greatly reduced.     

Human amniotic fluid promotes retinal pigmented epithelial cells' trans-differentiation into rod photoreceptors and retinal ganglion cells

To evaluate the effect of human amniotic fluid (HAF) on retinal pigmented epithelial cells growth and trans-differentiation into retinal neurons, retinal pigmented epithelium (RPE) cells were isolated from neonatal human cadaver eye globes and cultured in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using FBS-containing or HAF-containing media. Amniotic fluid samples were received from pregnant women in the first trimester of gestation. Cell proliferation and death enzyme-linked immunosorbent assays were performed to assess the effect of HAF on RPE cell growth. Trans-differentiation into rod photoreceptors and retinal ganglion cells was also studied using immunocytochemistry and real-time polymerase chain reaction techniques. Primary cultures of RPE cells were successfully established under FBS-containing or HAF-containing media leading to rapid cell growth and proliferation. When RPE cells were moved to in vitro culture system, they began to lose their differentiation markers such as pigmentation and RPE65 marker and trans-differentiated neural-like cells followed by spheroid colonies pertaining to stem/progenitor cells were morphologically detected. Immunocytochemistry (ICC) analysis of HAF-treated cultures showed a considerable expression of Rhodopsin gene (30% Rhodopsin-positive cells) indicating trans-differentiation of RPE cells to rod photoreceptors. Real-time polymerase chain reaction revealed an HAF-dose-dependant expression of Thy-1 gene (RGC marker) and significant promoting effect of HAF on RGCs generation. The data presented here suggest that HAF possesses invaluable stimulatory effect on RPE cells growth and trans-differentiation into retinal neurons. It can be regarded as a newly introduced enriched supplement in serum-free kinds of media used in neuro-retinal regeneration studies.     

Derivation of smooth muscle cells with neural crest origin from human induced pluripotent stem cells

The heterogeneity of vascular smooth muscle cells (SMCs) is related to their different developmental origins such as the neural crest and mesoderm. Derivation of SMCs from different origins will provide valuable in vitro models for the investigation of vascular development and diseases. From the perspective of regenerative medicine and tissue engineering, an expandable cell source of SMCs is required for the construction of tissue-engineered blood vessels. In this study, we developed a robust protocol to derive neural crest stem cells (NCSCs) from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). NCSCs derived from ESCs and iPSCs were expandable with similar cell doubling times. NCSCs were capable of differentiating into neural and mesenchymal lineages. TGF-β1 induced the expression of SMC markers calponin-1, SM22α, and smooth muscle myosin heavy chain and resulted in the assembly of smooth muscle α-actin, calponin-1, and SM22α into stress fibers. This work provides a basis for using iPSCs to study SMC biology and deriving vascular cells for tissue engineering.     

bFGF诱导人胚视网膜色素上皮细胞转分化的研究

目的 探讨bFGF体外诱导早期人胚视网膜色素上皮（RPE）细胞转分化现象及其分子机制,为视网膜发育的进一步基础研究和临床RPE细胞移植奠定基础.方法 取4～6代人胚（6 w～10 w）RPE细胞,bFGF（20 ng/ml）诱导培养（4 d～6 d）后观察RPE细胞形态学的改变,用免疫细胞化学作转分化RPE细胞鉴定.RT-PCR分析转分化RPE细胞小眼畸形基因（MITF）的表达.结果 bFGF诱导培养后的人胚RPE细胞呈现类神经样细胞表型,不仅表达自身上皮细胞标记物角蛋白（Keratin）,同时还表达多种神经视网膜上皮细胞标记物（MAP2、GS、GFAP、Peripherin、Opsin,但不表达NCAM）;用RT-PCR方法没有检测到转分化RPE细胞MITF-A mRNA表达.结论 早期人胚RPE细胞的发育具有一定的可塑性,bFGF可以诱导早期人胚RPE细胞表达多种神经视网膜上皮细胞标记物,可能是通过抑制MITF-A的分子信号通路.