Cell Differentiation Protocols Research Articles

When rejuvenation is a problem: challenges of modeling late-onset neurodegenerative disease

In contrast to the successful modeling of early-onset disorders using patient-specific cells, modeling of late-onset neurodegenerative diseases such as Parkinson's disease remains a challenge. This might be related to the often ignored fact that current induced pluripotent stem cell (iPSC) differentiation protocols yield cells that typically show the behavior of fetal stage cells. Acknowledging aging as a contributing factor in late-onset neurodegenerative disorders represents an important step on the road towards faithfully recreating these diseases in vitro. Here, we summarize progress in the field and review the strategies and challenges for triggering late-onset disease phenotypes.

Inducible Gata1 suppression expands megakaryocyte-erythroid progenitors from embryonic stem cells.

Transfusion of donor-derived platelets is commonly used for thrombocytopenia, which results from a variety of clinical conditions and relies on a constant donor supply due to the limited shelf life of these cells. Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies; however, the majority of current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. In both mice and humans, mutations in the gene-encoding transcription factor GATA1 cause an accumulation of proliferating, developmentally arrested megakaryocytes, suggesting that GATA1 suppression in ES and iPS cell-derived hematopoietic progenitors may enhance megakaryocyte production. Here, we engineered ES cells from WT mice to express a doxycycline-regulated (dox-regulated) shRNA that targets Gata1 transcripts for degradation. Differentiation of these cells in the presence of dox and thrombopoietin (TPO) resulted in an exponential (at least 10¹³-fold) expansion of immature hematopoietic progenitors. Dox withdrawal in combination with multilineage cytokines restored GATA1 expression, resulting in differentiation into erythroblasts and megakaryocytes. Following transfusion into recipient animals, these dox-deprived mature megakaryocytes generated functional platelets. Our findings provide a readily reproducible strategy to exponentially expand ES cell-derived megakaryocyte-erythroid progenitors that have the capacity to differentiate into functional platelet-producing megakaryocytes.

Concise review: growing hearts in the right place: on the design of biomimetic materials for cardiac stem cell differentiation.

Tissue engineering aims at recapitulating permissive conditions that enable cells to collaborate and form functional tissues. Applications range from human tissue modeling for diagnostic purposes to therapeutic solutions in regenerative medicine and surgery. Across this spectrum, human stem cells are the active ingredient, expandable virtually indefinitely and with the propensity to generate new tissue. Engaging lineage-specific differentiation requires a precise concerto of key spatial and temporal factors, such as soluble molecules and growth factors, but also physical and mechanical stimuli. These stimuli compete to modulate distinct developmental signaling pathways and ultimately affect the differentiation efficiency. The heart is a chemo-mechano-electrical biological system that behaves as both a sensor and an actuator. It can transduce electrical inputs to generate mechanical contraction and electrical wave propagation. Such a complex organ arises from multipart developmental events that interact with one another to self-regulate. Here, we overview the main events of heart development and the role of mechanical forces in modifying the microenvironment of the progenitor cells. We analyze the cascades regulating cardiac gene activation to illustrate how mechanotransduction is already involved in the most popular protocols for stem cell differentiation (SCD) into cardiomyocytes. We then review how forces are transmitted to embryonic stem cells by cell-substrate or cell-cell communications, and how biomaterials can be designed to mimic these interactions and help reproduce key features of the developmental milieu. Putting this back in a clinical perspective, many challenges need to be overcome before biomaterials-based SCD protocols can be scaled up and marketed.

Arterial specification of endothelial cells derived from human induced pluripotent stem cells in a biomimetic flow bioreactor

Endothelial cells (ECs) exist in different microenvironments in vivo, including under different levels of shear stress in arteries versus veins. Standard stem cell differentiation protocols to derive ECs and EC-subtypes from human induced pluripotent stem cells (hiPSCs) generally use growth factors or other soluble factors in an effort to specify cell fate. In this study, a biomimetic flow bioreactor was used to subject hiPSC-derived ECs (hiPSC-ECs) to shear stress to determine the impacts on phenotype and upregulation of markers associated with an anti-thrombotic, anti-inflammatory, arterial-like phenotype. The in vitro bioreactor system was able to efficiently mature hiPSC-ECs into arterial-like cells in 24 h, as demonstrated by qRT-PCR for arterial markers EphrinB2, CXCR4, Conexin40 and Notch1, as well protein-level expression of Notch1 intracellular domain (NICD). Furthermore, the exogenous addition of soluble factors was not able to fully recapitulate this phenotype that was imparted by shear stress exposure. The induction of these phenotypic changes was biomechanically mediated in the shear stress bioreactor. This biomimetic flow bioreactor is an effective means for the differentiation of hiPSC-ECs toward an arterial-like phenotype, and is amenable to scale-up for culturing large quantities of cells for tissue engineering applications.

Enrichment of human embryonic stem cell derived motor neuron cultures using arabinofuranosyl cytidine

ABSTRACT Aim: Human embryonic stem cell (hESC)-derived motor neurons (MNs) can model neurodegenerative diseases, in other words, for the development of cell-based drug screening assays. However, efficient MN generation is challenging and high level of contamination of undifferentiated cells remains a significant problem when culturing hESC-derived MNs. We describe a protocol for the removal of undifferentiated cells using the DNA synthesis inhibitor arabinofuranosyl cytidine (Ara-C). Materials & methods: hESC-MNs were treated with Ara-C after the last step of differentiation. FACS analysis and fluorescence microscopy were used to identify and quantify MNs. Results: HB9/ChAT-positive live mature MNs were enriched 3.9-times in Ara-C-treated cultures when compared with untreated cells. The Ara-C-treated MNs are electrophysiologically functional and discharge action potentials. Conclusion: Ara-C selection of MNs can be combined with stem cell differentiation protocols to enrich MNs in culture.

In Vitro Differentiation of Pluripotent Stem Cells into Functional β Islets Under 2D and 3D Culture Conditions and In Vivo Preclinical Validation of 3D Islets.

Since the advent of pluripotent stem cells, (embryonic and induced pluripotent stem cells), applications of such pluripotent stem cells are of prime importance. Indeed, scientists are involved in studying the basic biology of pluripotent stem cells, but equal impetus is there to direct the pluripotent stem cells into multiple lineages for cell therapy applications. Scientists across the globe have been successful, to a certain extent, in obtaining cells of definitive endoderm and also pancreatic β islets by differentiating human pluripotent stem cells. Pluripotent stem cell differentiation protocols aim at mimicking in vivo embryonic development. As in vivo embryonic development is a complex process and involves interplay of multiple cytokines, the differentiation protocols also involve a stepwise use of multiple cytokines. Indeed the novel markers for pancreas organogenesis serve as the roadmaps to develop new protocols for pancreatic differentiation from pluripotent stem cells. Earliest developed protocols for pancreas differentiation involved "Nestin selection pathway," a pathway common for both neuronal and pancreatic differentiation lead to the generation of cells that were a combination of cells from neuronal lineage. Eventually with the discovery of hierarchy of β cell transcription factors like Pdx1, Pax4, and Nkx2.2, forced expression of such transcription factors proved successful in converting a pluripotent stem cell into a β cell. Protocols developed almost half a decade ago to the recent ones rather involve stepwise differentiations involving various cytokines and could generate as high as 25 % functional insulin-positive cells in vitro. Most advanced protocols for β islet differentiations from human pluripotent stem cells focused on 3D culture conditions, which reportedly produced 60-65 % functional β islet cells. Here, we describe the protocol for differentiation of human pluripotent stem cells into functional β cells under both 2D and 3D culture conditions.

Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell–derived medium spiny neurons

A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening.

Inducible Gata1 Suppression As a Novel Strategy to Expand Physiologic Megakaryocyte Production from Embryonic Stem Cells

Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent potential sources of megakaryocytes and platelets for transfusion therapy. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny, including platelet-releasing megakaryocytes. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. Previously, we reported that in vitro differentiation of Gata1-null murine ES cells generated self-renewing hematopoietic progenitors termed G1ME cells that differentiated into erythroblasts and megakaryocytes upon restoration of Gata1 cDNA by retroviral transfer. However, terminal maturation of Gata1-rescued megakaryocytes was aberrant with immature morphology and no proplatelet formation, presumably due to non-physiological expression of GATA1. We now engineered wild type (WT) murine ES cells that express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs to develop a strategy for Gata1-blockade that upon its release, restores physiologic GATA1 expression during megakaryopoiesis. In vitro hematopoietic differentiation of control scramble shRNA-expressing ES cells with dox and thrombopoietin (TPO) produced megakaryocytes that underwent senescence after 7 days. Under similar differentiation conditions, Gata1 shRNA-expressing ES cells produced immature hematopoietic progenitors, termed G1ME2 cells, which replicated continuously for more than 40 days, resulting in ~1013-fold expansion (N=4 separate experiments). Upon dox withdrawal with multi-lineage cytokines present (EPO, TPO, SCF, GMCSF and IL3), endogenous GATA1 expression was restored to G1ME2 cells followed by differentiation into erythroblasts and megakaryocytes, but no myeloid cells. In clonal methylcellulose assays, dox-deprived G1ME2 cells produced a mixture of erythroid, megakaryocytic and erythro-megakaryocytic colonies. In liquid culture with TPO alone, dox-deprived G1ME2 cells formed mature megakaryocytes in 5-6 days, as determined by morphology, ultrastructure, acetylcholinesterase staining, upregulated megakaryocytic gene expression (Vwf, Pf4, Gp1ba, Selp, Ppbp), CD42b surface expression, increased DNA ploidy and proplatelet production. Compared to G1ME cells rescued with Gata1 cDNA retrovirus, dox-deprived G1ME2 cells exhibited more robust megakaryocytic maturation, similar to that of megakaryocytes produced from cultured fetal liver. Importantly, G1ME2 cell-derived megakaryocytes generated proplatelets in vitro and functional platelets in vivo (~40 platelets/megakaryocyte with a circulating half life of 5-6 hours). These platelets were actively incorporated into growing arteriolar thrombi at sites of laser injury and subsequently expressed the platelet activation marker p-selectin (N=3-4 separate experiments). Our findings indicate that precise timing and magnitude of a transcription factor is required for proper terminal hematopoiesis. We illustrate this principle using a novel, readily reproducible strategy to expand ES cell-derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes and then into functional platelets in clinically relevant numbers. DisclosuresNo relevant conflicts of interest to declare.

Differentiation of pluripotent stem cells into striatal projection neurons: a pure MSN fate may not be sufficient.

Huntington’s disease (HD) is an autosomal dominant inherited disorder leading to the loss inter alia of DARPP-32 positive medium spiny projection neurons (“MSNs”) in the striatum. There is no known cure for HD but the relative specificity of cell loss early in the disease has made cell replacement by neural transplantation an attractive therapeutic possibility. Transplantation of human fetal striatal precursor cells has shown “proof-of-principle” in clinical trials; however, the practical and ethical difficulties associated with sourcing fetal tissues have stimulated the need to identify alternative source(s) of donor cells that are more readily available and more suitable for standardization. We now have available the first generation of protocols to generate DARPP-32 positive MSN-like neurons from pluripotent stem cells and these have been successfully grafted into animal models of HD. However, whether these grafts can provide stable functional recovery to the level that can regularly be achieved with primary fetal striatal grafts remains to be demonstrated. Of particular concern, primary fetal striatal grafts are not homogenous; they contain not only the MSN subpopulation of striatal projection neurons but also include all the different cell types that make up the mature striatum, such as the multiple populations of striatal interneurons and striatal glia, and which certainly contribute to normal striatal function. By contrast, present protocols for pluripotent stem cell differentiation are almost entirely targeted at specifying just neurons of an MSN lineage. So far, evidence for the functionality and integration of stem-cell derived grafts is correspondingly limited. Indeed, consideration of the features of full striatal reconstruction that is achieved with primary fetal striatal grafts suggests that optimal success of the next generations of stem cell-derived replacement therapy in HD will require that graft protocols be developed to allow inclusion of multiple striatal cell types, such as interneurons and/or glia. Almost certainly, therefore, more sophisticated differentiation protocols will be necessary, over and above replacement of a specific population of MSNs. A rational solution to this technical challenge requires that we re-address the underlying question—what constitutes a functional striatal graft?

Important Topics in the Future of Tissue Engineering: Comments from the participants of the 5th International Conference on Tissue Engineering at Kos, Greece.

Over 150 participants from around the world congregated on the beautiful island of Kos—home of Hippocrates, Father of Medicine—from June 20 to June 25, 2014, to share their research findings and engage in dialogue pertaining to progress in tissue engineering. Since the field’s inception, we have witnessed its astonishing development over the past 30 years and the groundbreaking work stemming from both laboratory and clinical settings. Despite continuously evolving concepts and strategies, the essential ingredients of scaffolds, cells and growth factors remain to be explored further. While stem cells presently pique tremendous interest within the research community, they have yet to come to the mainstream in the clinical context, as current regulated medical devices predominantly involve only scaffolds and several growth factors. In light of this bench-to-bedside disparity, participants should evaluate the translational aspects of their work and how it can be incorporated into the future of tissue engineering and regenerative medicine. Furthermore, researchers may consider expanding their focus to include currently untapped technologies with potential to propel the field forward. The 5th International Conference on Tissue Engineering provided a global forum for established leaders and emerging young scientists in the field, to showcase their diverse ideas on advanced tissue engineering and regenerative medicine. In particular, new biomaterial-based strategies were proposed to improve the quality of engineered tissue, to increase spatiotemporal release of growth factors, and to elucidate and modulate cell signaling pathways. Improvements in scaffold fabrication methodologies, cell delivery approaches and cell differentiation protocols were also discussed. The topical breadth was further demonstrated through presentations on imaging modalities and computational modeling. Overall, this conference provided a comprehensive, enriching overview of the field and alluded to trends in the years to come. In what follows, we present a commentary from nine participants who are active professors representing various branches of tissue engineering. We hope that their insight may stimulate further discussion by the readers.

Regeneration of uterine horns in rats using collagen scaffolds loaded with human embryonic stem cell-derived endometrium-like cells.

A variety of diseases may lead to hysterectomies or uterine injuries, which may form a scar and lead to infertility. Due to the limitation of native materials, there are a few effective methods to treat such damages. Tissue engineering combines cell and molecular biology with materials and mechanical engineering to replace or repair damaged organs and tissues. The use of human embryonic stem cells (hESCs) as a donor cell source for the replacement therapy will require the development of simple and reliable cell differentiation protocols. This study aimed at efficiently generating endometrium-like cells from the hESCs and at using these cells with collagen scaffold to repair uterine damage. The hESCs were induced by co-culturing with endometrial stromal cells, and simultaneously added cytokines: epidermal growth factor (EGF), platelet-derived growth factor-b (PDGF-b), and E2. Expression of cell specific markers was analyzed by immunofluorescence and reverse trascription-polymerase chain reaction to monitor the progression toward an endometrium-like cell fate. After differentiation, the majority of cells (>80%) were positive for cytokeratin-7, and the expression of key transcription factors related to endometrial development, such as Wnt4, Wnt7a, Wnt5a, Hoxa11, and factors associated with endometrial epithelial cell function: Hoxa10, Intergrinβ3, LIF, ER, and PR were also detected. Then, we established the uterine full-thickness-injury rat models to test cell function in vivo. hESC-derived cells were dropped onto collagen scaffolds and transplanted into the animal model. Twelve weeks after transplantation, we discovered that the hESC-derived cells could survive and recover the structure and function of uterine horns in a rat model of severe uterine damage. The experimental system presented here provides a reliable protocol to produce endometrium-like cells from hESCs. Our results encourage the use of hESCs in cell-replacement therapy for severe uterine damage in future.

Zinc-finger nuclease enhanced gene targeting in human embryonic stem cells.

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations. These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes. This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered. However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient. This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson's disease modeling or cell replacement therapy.

Abstract 142: Defining the Non-Myocyte Compartment is Key for Enhanced Maturation of Human Engineered Heart Muscle

Background: Tissue engineering of heart muscle from human pluripotent stem cells holds great potential for in vitro studies, disease modeling, and cardiac replacement therapy. A number of variables may however affect maturation and function of human cardiomyocytes (CM) in tissue engineered heart muscle (EHM). Here, we hypothesized that defined non-myocyte (NM) populations support structural and functional maturation of EHM. Methods and Results: To investigate the role of non-myocytes (NM) for heart muscle assembly in vitro we generated EHM from purified CM (93±1.5% actinin+) and a mixture of CM and NM (70/30%). Notably, only the NM-supplemented EHM generated measurable forces (0.8±0.1 mN, n=9) with anisotropically aligned cardiomyocytes. Depending on pluripotent stem cell line and differentiation protocol the NM compartment may vary considerably. To further define the influence of the NM compartment we generated EHM from HES2-derived CM with undefined NM, i.e the NM typically derived during cardiac differentiation, and defined NM (fibroblasts). Defined EHM were more mature with higher forces and lower variability between experimental series (defined: 9.8±0.9 nN/CM, undefined: 4.7±1.4 nN/CM, n=10/9), higher EC50 for calcium, and enhanced inotropic response to isoprenaline despite comparable CM:NM composition of 1:1. Increased actinin protein per CM, a reduction of MLC2V/2A double positive CM, and evidence of CM cycle withdrawal indicated enhanced ventricular maturation in defined EHM. Next, we tested whether defining cell composition and NM in iPS-derived EHM will yield a comparable functional phenotype to HES2-EHM. In agreement with the above data, defined iPS-EHM displayed advanced functional maturation with high specific forces, comparable calcium EC50, and inotropic response to isoprenaline. Summary and Conclusions: Here we demonstrate that defining the NM compartment is essential for optimized human heart muscle formation and maturation in vitro. Moreover, our data provide (1) evidence for the applicability of EHM in modelling of heart muscle development and (2) a strong rationale for the need to define CM and NM compartments in tissue engineered myocardium to reduce variability in applications such as disease modelling.

A multiplex high-throughput gene expression assay to simultaneously detect disease and functional markers in induced pluripotent stem cell-derived retinal pigment epithelium.

There is continuing interest in the development of lineage-specific cells from induced pluripotent stem (iPS) cells for use in cell therapies and drug discovery. Although in most cases differentiated cells show features of the desired lineage, they retain fetal gene expression and do not fully mature into "adult-like" cells. Such cells may not serve as an effective therapy because, once implanted, immature cells pose the risk of uncontrolled growth. Therefore, there is a need to optimize lineage-specific stem cell differentiation protocols to produce cells that no longer express fetal genes and have attained "adult-like" phenotypes. Toward that goal, it is critical to develop assays that simultaneously measure cell function and disease markers in high-throughput format. Here, we use a multiplex high-throughput gene expression assay that simultaneously detects endogenous expression of multiple developmental, functional, and disease markers in iPS cell-derived retinal pigment epithelium (RPE). We optimized protocols to differentiate iPS cell-derived RPE that was then grown in 96- and 384-well plates. As a proof of principle, we demonstrate differential expression of eight genes in iPS cells, iPS cell-derived RPE at two different differentiation stages, and primary human RPE using this multiplex assay. The data obtained from the multiplex gene expression assay are significantly correlated with standard quantitative reverse transcription-polymerase chain reaction-based measurements, confirming the ability of this high-throughput assay to measure relevant gene expression changes. This assay provides the basis to screen for compounds that improve RPE function and maturation and target disease pathways, thus providing the basis for effective treatments of several retinal degenerative diseases.

Differentiation of human pluripotent stem cells into nephron progenitor cells in a serum and feeder free system.

ObjectivesKidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney, stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources, pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However, little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study, we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system.Materials and MethodsWe designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak, intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR, real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility.ResultsAfter modification of culture period and concentration of exogenous factors, hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2, GDNF, HOXD11, WT1 and CITED1 in addition to OSR1, PAX2, SALL1 and EYA1. Moreover, NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular, approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems.ConclusionsOur novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases.

Pancreas development in humans

We highlight some of the major recent advances in characterizing human pancreas development and endocrine cell differentiation. Extensive research efforts have helped to define crucial events in the mouse pancreas organogenesis. Information gained from these studies was used to develop human embryonic stem cell (hESC) differentiation protocols with the goal of generating functional glucose-responsive, insulin-producing human β-cells. In spite of remarkable progress in hESC differentiation, current protocols based on mouse developmental biology can produce human β-cells only in vivo. New differentiation markers and recently generated reagents may provide an unprecedented opportunity to develop a high-density expression map of human fetal pancreas and pancreatic islets that could serve as a reference point for in vitro hESC differentiation. Integrating an increased knowledge of human pancreas development into hESC differentiation protocols has the potential to greatly advance our ability to generate functional insulin-producing cells for β-cell replacement therapy.

Comparison of different protocols for neural differentiation of human induced pluripotent stem cells

Although embryonic stem cells (ESCs) have enormous potentials due to their pluripotency, their therapeutic use is limited by ethical, biological and safety issues. Compared to ESCs, induced pluripotent stem cells (iPSCs) can be obtained from mouse or human fibroblasts by reprogramming. Numerous studies have established many protocols for differentiation of human iPSCs (hiPSCs) into neural lineages. However, the low differentiation efficiency of such protocols motivates researchers to design new protocols for high yield differentiation. Herein, we compared neural differentiation potential of three induction media for conversion of hiPSCs into neural lineages. In this study, hiPSCs-derived embryoid bodies were plated on laminin coated dishes and were treated with three induction media including (1) bFGF, EGF (2) RA and (3) forskolin, IBMX. Immunofluorescence staining and quantitative real-time PCR (qPCR) analysis were used to detect the expression of neural genes and proteins. qPCR analysis showed that the expression of neural genes in differentiated hiPSCs in forskolin, IBMX supplemented media was significantly higher than undifferentiated cells and those in induction media containing bFGF, EGF or RA. In conclusion, our results indicated a successful establishment protocol with high efficiency for differentiation of hiPSCs into neural lineages.

Inhibition of stearoyl-coA desaturase selectively eliminates tumorigenic Nanog-positive cells: Improving the safety of iPS cell transplantation to myocardium

Induced pluripotent stem cells (iPS) can differentiate into cardiomyocytes (CM) and represent a promising form of cellular therapy for heart regeneration. However, residual undifferentiated iPS derivates (iPSD), which are not fully eliminated by cell differentiation or purification protocols, may form tumors after transplantation, thus compromising therapeutic application. Inhibition of stearoyl-coA desaturase (SCD) has recently been reported to eliminate undifferentiated human embryonic stem cells, which share many features with iPSD. Here, we tested the effects of PluriSin#1, a small-molecule inhibitor of SCD, on iPS-derived CM. We found that plurisin#1 treatment significantly decreased the mRNA and protein level of Nanog, a marker for both cell pluripotency and tumor progression; importantly, we provide evidence that PluriSin#1 treatment at 20 µM for 1 day significantly induces the apoptosis of Nanog-positive iPSD. In addition, PluriSin#1 treatment at 20 µM for 4 days diminished Nanog-positive stem cells in cultured iPSD while not increasing apoptosis of iPS-derived CM. To investigate whether PluriSin#1 treatment prevents tumorigenicity of iPSD after cell transplantation, we intramyocardially injected PluriSin#1- or DMSO-treated iPSD in a mouse model of myocardial infarction (MI). DMSO-treated iPSD readily formed Nanog-expressing tumors 2 weeks after injection, which was prevented by treatment with PluriSin#1. Moreover, treatment with PluriSin#1 did not change the expression of cTnI, α-MHC, or MLC-2v, markers of cardiac differentiation (P > 0.05, n = 4). Importantly, pluriSin#1-treated iPS-derived CM exhibited the ability to engraft and survive in the infarcted myocardium. We conclude that inhibition of SCD holds the potential to enhance the safety of therapeutic application of iPS cells for heart regeneration.

Fare Embriyonik Kök Hücrelerden Nöronal Hücrelerin Farklılaşması

With new technologies emerging today, the importance of stem cells in the cell therapy of nervous system diseases is supported by recent studies. Therefore, the development of neuronal cell differentiation protocols from stem cells is of great importance. In our study, the differentiation of neuronal and neuroglial cells from mouse embryonic stem (ES) cell line and their analysis with neuronal cell markers are aimed. Mouse ES cells were differentiated to neurogenic series cells by adding N2 and bFGF to the culture medium on coated Fibronectin dishes. For the identification of differentiated cells, they were evaluated by light microscopy using immunhistochemistry techniques and by electron microscopy. Indirect immunohistochemical staining method was performed with SSEA-1 (mouse embriyonic stem cells marker), Nestin (neural precursor cells marker), βIII-Tubulin (neuronal cells marker), MAP-2 (neuronal cells marker), GFAP (astrocyte marker), and O4 (oligodendrocyte marker). After 1 week of differentiation of cells, immunoreactivities of SSEA-1 and Nestin were detected to be negative and moderate, respectively. After 2 weeks culture time, the differentiation was still continuing and especially positive immunoreactivities of β-III Tubulin and MAP-2 and weak immunoreactivities of O4 and GFAP were supported neuronal differentiation. In conclusion, our results suggest that neuronal cell derived from mouse ES cells were differentiated particularly to neuron using N2+bFGF+fibronectin culture condition. Therefore, these differentiated cells may be used as a treatment method in degenerative diseases of the nervous system.

How important is differentiation in the therapeutic effect of mesenchymal stromal cells in liver disease?

Background aimsThe protocols for differentiation of hepatocyte-like cells (HLCs) from mesenchymal stromal cells (MSCs) have been well established. Previous data have shown that MSCs and their derived HLCs were able to engraft injured liver and alleviate injuries induced by carbon tetrachloride. The goal of the current study was to determine the differences of MSCs and their derived HLCs in terms of therapeutic functions in liver diseases. MethodsAfter hepatic differentiation of umbilical cord–derived MSCs in vitro, we detected both MSC and HLC expressions of adhesion molecules and chemokine receptor CXCR4 by flow cytometry; immunosuppressive potential and hepatocyte growth factor expression were determined by means of enzyme-linked immunosorbent assay. We compared the therapeutic effect for fulminant hepatic failure in a mouse model. ResultsMSC-derived-HLCs expressed lower levels of hepatocyte growth factor, accompanied by impaired immunosuppression in comparison with MSCs. Furthermore, undifferentiated MSCs showed rescuing potentials superior to those in HLCs for the treatment of fulminant hepatic failure. ConclusionsAfter differentiation, HLCs lost several major properties in comparison with undifferentiated MSCs, which are beneficial for their application in liver diseases. Undifferentiated MSCs may be more appropriate than are HLCs for the treatment of liver diseases.