Retinoic acid promotes neural conversion of mouse embryonic stem cells in adherent monoculture

Retinoic acid (RA) plays multiple roles in the nervous system, including induction of neural differentiation, axon outgrowth and neural patterning. Previously, RA for neural differentiation of embryonic stem (ES) cells always relies on embryoid bodies (EBs) formation. Here we report an in vitro adherent monoculture system to induce mouse ES cells into neural cells accompanied with RA. RA (1 μM) treatment, during initial 2 days of differentiation, can enhance the expression of neural markers, such as Nestin, Tuj1 and MAP2, and result in an earlier neural differentiation of ES cells. Furthermore, RA promotes a significant increase in neurite elongation of ES-derived neurons. Our study also implies that RA induced to express Wnt antagonist Dickkopf-1 (Dkk-1) for neural differentiation. However, the mechanisms of RA triggering neural induction remain to be determined. Our simple and efficient strategy is proposed to provide a basis for studying RA signaling pathways in neural differentiation in vitro.     

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.     

Comparison of long-term retinoic acid-based neural induction methods of bone marrow human mesenchymal stem cells

The differentiation of human mesenchymal stem cells (hMSCs) into neural cells in vitro provides a potential tool to be utilized for cell therapy of neurodegenerative disorders. Although previous studies repeated different protocols for the induction of neural cells from hMSCs in vitro, the results were not in complete agreement. In this study, we have attempted to compare three of these neural induction methods; retinoic acid (RA) treatment, RA treatment in serum reduced conditions, and treatment using other chemical compounds (dimethyl sulfoxide and potassium chloride) along with RA by real-time cell analysis and immunofluorescent staining of neural markers. RA treatment led to a slow progression of cells into neural-like morphology with the expression of neural protein neurofilament whereas reducing serum during RA treatment caused a much more extended differentiation process. Additionally, neural-like morphology was persistent in the later periods of differentiation in RA treatment. On the other hand, chemical induction caused cell shrinkages mimicking neural-like morphology in a short time and loss of this morphology along with increased cell death in later periods. Among the three methods compared, RA treatment was the most reliable one in terms of stability of differentiation and neural protein expressions.     

The adult human brain harbors multipotent perivascular mesenchymal stem cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.     

Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells

Retinoic acid (RA) is one of the most important morphogens, and its embryonic distribution correlates with neural differentiation and positional specification in the developing central nervous system. To investigate the concentration-dependent effects of RA on neural differentiation of mouse embryonic stem cells (ES cells), we investigated the precise expression profiles of neural and regional specific genes by ES cells aggregated into embryoid bodies (EBs) exposed to various concentrations of RA or the BMP antagonist Noggin. RA promoted both neural differentiation and caudalization in a concentration-dependent manner, and the concentration of RA was found to regulate dorso-ventral identity, i.e., higher concentrations of RA induced a dorsal phenotype, and lower concentrations of RA induced a more ventral phenotype. The induction of the more ventral phenotype was due to the higher expression level of the N-terminus of sonic hedgehog protein (Shh-N) when treated with low concentration RA, as it was abrogated by an inhibitor of Shh signaling, cyclopamine. These findings suggest that the concentration of RA strictly and simultaneously regulates the neuralization and positional specification during differentiation of mouse ES cells and that it may be possible to use it to establish a strategy for controlling the identity of ES-cell-derived neural cells.     

pp60src tyrosine kinase modulates P19 embryonal carcinoma cell fate by inhibiting neuronal but not epithelial differentiation

P19 embryonal carcinoma cells provide an in vitro model system to analyze the events involved in neural differentiation. These multipotential stem cells can be induced by retinoic acid (RA) to differentiate into neural cells. We have investigated the ability of several variant forms of the protein-tyrosine kinase (PTK) pp60src to modulate cell fate determination in this system. Normally, P19 cells are induced to differentiate along a neural lineage when allowed to form extensive cell-cell contacts in large multicellular aggregates during exposure to RA. Through analysis of markers of epithelial (keratin and desmosomal proteins) and neuronal (neurofilament) cells we have found that RA-induced P19 cells transiently express epithelial markers before neuronal differentiation. Under these inductive conditions, expression of pp60v-src or expression of the neuronal variant pp60c-src+ inhibited neuronal differentiation, and resulted in maintained expression of an epithelial phenotype. Morphological analysis showed that expression of pp60src PTKs results in decreased cell-cell adhesion during the critical cell aggregation stage of the neural differentiation procedure. The effects of pp60v-src on cell fate and cell-cell adhesion could be mimicked by direct modulation of Ca+(+)-dependent cell-cell contact during RA induction of normal P19 cells. We conclude that the neural lineage of P19 cells includes an early epithelial intermediate and suggest that tyrosine phosphorylation can modulate cell fate determination during an early cell-cell adhesion-dependent event in neurogenesis.     

Cell aggregation-induced FGF8 elevation is essential for P19 cell neural differentiation

FGF8, a member of the fibroblast growth factor (FGF) family, has been shown to play important roles in different developing systems. Mouse embryonic carcinoma P19 cells could be induced by retinoic acid (RA) to differentiate into neuroectodermal cell lineages, and this process is cell aggregation dependent. In this report, we show that FGF8 expression is transiently up-regulated upon P19 cell aggregation, and the aggregation-dependent FGF8 elevation is pluripotent stem cell related. Overexpressing FGF8 promotes RA-induced monolayer P19 cell neural differentiation. Inhibition of FGF8 expression by RNA interference or blocking FGF signaling by the FGF receptor inhibitor, SU5402, attenuates neural differentiation of the P19 cell. Blocking the bone morphogenetic protein (BMP) pathway by overexpressing Smad6 in P19 cells, we also show that FGF signaling plays a BMP inhibition-independent role in P19 cell neural differentiation.     

Penetration and differentiation of cephalic neural crest‐derived cells in the developing mouse telencephalon

Neural crest (NC) cells originate from the neural folds and migrate into the various embryonic regions where they differentiate into multiple cell types. A population of cephalic neural crest‐derived cells (NCDCs) penetrates back into the developing forebrain to differentiate into microvascular pericytes, but little is known about when and how cephalic NCDCs invade the telencephalon and differentiate into pericytes. Using a transgenic mouse line in which NCDCs are genetically labeled with enhanced green fluorescent protein (EGFP), we observed that NCDCs started to invade the telencephalon together with endothelial cells from embryonic day (E) 9.5. A majority of NCDCs located in the telencephalon expressed pericyte markers, that is, PDGFRβ and NG2, and differentiated into pericytes around E11.5. Surprisingly, many of the NC‐derived pericytes express p75, an undifferentiated NCDC marker at E11.5, as well as NCDCs in the mesenchyme. At the same time, a minor population of NCDCs that located separately from blood vessels in the telencephalon were NG2‐negative and some of these NCDCs also expressed p75. Proliferation and differentiation of pericytes appeared to occur in a specific mesenchymal region where blood vessels penetrated into the telencephalon. These results indicate that (i) NCDCs penetrate back into the telencephalon in parallel with angiogenesis, (ii) many NC‐derived pericytes may be still in pre‐mature states even though after differentiation into pericytes in the early developing stages, (iii) a small minority of NCDCs may retain undifferentiated states in the developing telencephalon, and (iv) a majority of NCDCs proliferate and differentiate into pericytes in the mesenchyme around the telencephalon.     

Embryonic stem cells as a model for studying regulation of cellular differentiation

Mouse embryonic stem (ES) cells can be differentiated in vitro into near homogeneous populations of both neurons and skeletal muscle as well as other cell types. We previously showed that treatment of pluripotent ES cells with retinoic acid (RA) induced differentiation into highly enriched populations of gamma-aminobutyric acid (GABA) expressing neurons. The reasons for generation of only GABA neurons as opposed to other neuronal cell types were not known. We have extended our previous work and now show that with RA induction of ES cells we not only obtain GABA neurons, but also dopaminergic neurons. Critical for the production of dopaminergic neurons after RA induction was the post-induction plating conditions used. No dopaminergic neurons were detected if cells were plated in serum-free media optimized for neuronal survival. However, significant numbers of dopamine neurons could be detected when cells were plated in media containing fetal calf serum. These observations support the conclusion that RA acts as a general neural inducing agent and that conditions post-induction either selectively support survival of a particular class of neuronal cells or that the conditions post-induction actually further instruct cells to differentiate into different types of neurons.     

The impact of pericytes on the blood-brain barrier integrity depends critically on the pericyte differentiation stage

The blood-brain barrier consists of the cerebral microvascular endothelium, pericytes, astrocytes and neurons. In this study we analyzed the differentiation stage dependent influence of primary porcine brain capillary pericytes on the barrier integrity of primary porcine brain capillary endothelial cells. At first, we were able to induce two distinct differentiation stages of the primary pericytes in vitro. TGFβ treated pericytes expressed more α-SMA and actin while desmin, vimentin and nestin expression was decreased when compared to bFGF induced cells. Further analysis of α-SMA revealed that most of the pericytes differentiated with TGFβ expressed functional α-SMA while only few cells expressed functional α-SMA in the presence of bFGF. In addition the permeability factors VEGF, MMP-2 and MMP-9 were higher secreted by the α-SMA positive phenotype indicating a proangiogenic role of this TGFβ induced pericyte differentiation stage. Higher level of VEGF, MMP-2 and MMP-9 were as well detected in the TGFβ pretreated pericyte coculture with endothelial cells when compared to the influence of the bFGF pretreated pericytes. The TEER measurement of the barrier integrity of endothelial cells revealed that bFGF induced α-SMA negative pericytes stabilize the barrier integrity while α-SMA positive pericytes differentiated by TGFβ decrease the barrier integrity. These results together reveal the potential of pericytes to regulate the endothelial barrier integrity in a differentiation stage dependant pathway.     

Misexpression of mouse porcupine isoforms modulates the differentiation of P19 embryonic carcinoma cells

Drosophila porcupine (porc) encodes an ER membrane protein that is required for the processing of the Drosophila Wnt family. Homologs of porc have been identified in various multicellular organisms and have been implicated in the biosynthesis of Wnt proteins. In contrast to Drosophila, vertebrates generate four different porc mRNAs (A-D) by alternative splicing. Murine porcD (MporcD) mRNA levels transiently increase during the neuroectodermal differentiation of P19 cells, but diminish during mesodermal differentiation. P19 cells constitutively expressing mouse porcA (MporcA), but not MporcD, undergo apoptosis by the induction of neuroectodermal differentiation. Meanwhile, P19 cells constitutively expressing MporcD, but not MporcA, do not adopt mesodermal cell morphology and fail to express myf-5 when induced to mesodermal differentiation. These results therefore demonstrate that the alternative splicing of Mporc is regulated in a cell-type specific manner, and the resulting Mporc isoforms have different functions in the neuroectodermal and mesodermal differentiation of P19 cells.     

Neural differentiation of pluripotent mouse embryonal carcinoma cells by retinoic acid: inhibitory effect of serum

In both embryonal carcinoma (EC) and embryonic stem (ES) cells, the differentiation pathway entered after treatment with retinoic acid (RA) varies as it is based upon different conditions of culture. This study employs mouse EC cells P19 to investigate the effects of serum on RA-induced neural differentiation occurring in a simplified monolayer culture. Cell morphology and expression of lineage-specific molecular markers document that, while non-neural cell types arise after treatment with RA under serum-containing conditions, in chemically defined serum-free media RA induces massive neural differentiation in concentrations of 10(-9) M and higher. Moreover, not only neural (Mash-1) and neuroectodermal (Pax-6), but also endodermal (GATA-4, alpha-fetoprotein) genes are expressed at early stages of differentiation driven by RA under serum-free conditions. Furthermore, as determined by the luciferase reporter assay, the presence or absence of the serum does not affect the activity of the retinoic acid response element (RARE). Thus, mouse EC cells are able to produce neural cells upon exposure to RA even without culture in three-dimensional embryoid bodies (EBs). However, in contrast to standard EBs-involving protocol(s), neural differentiation in monolayer only takes place when complex signaling from serum factors is avoided. This simple and efficient strategy is proposed to serve as a basis for neurodifferentiation studies in vitro.     

Efficient differentiation of mouse embryonic stem cells into motor neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test). Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.     

Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells.

In this report, we show that the overexpression of tissue transglutaminase (tTG) in the human neuroblastoma cell line SK-N-BE(2) renders these neural crest-derived cells highly susceptible to death by apoptosis. Cells transfected with a full-length tTG cDNA, under the control of a constitutive promoter, show a drastic reduction in proliferative capacity paralleled by a large increase in cell death rate. The dying tTG-transfected cells exhibit both cytoplasmic and nuclear changes characteristic of cells undergoing apoptosis. The tTG-transfected cells express high Bcl-2 protein levels as well as phenotypic neural cell adhesion molecule markers (NCAM and neurofilaments) of cells differentiating along the neuronal pathway. In keeping with these findings, transfection of neuroblastoma cells with an expression vector containing segments of the human tTG cDNA in antisense orientation resulted in a pronounced decrease of both spontaneous and retinoic acid (RA)-induced apoptosis. We also present evidence that (i) the apoptotic program of these neuroectodermal cells is strictly regulated by RA and (ii) cell death by apoptosis in the human neuroblastoma SK-N-BE(2) cells preferentially occurs in the substrate-adherent phenotype. For the first time, we report here a direct effect of tTG in the phenotypic maturation toward apoptosis. These results indicate that the tTG-dependent irreversible cross-linking of intracellular protein represents an important biochemical event in the induction of the structural changes featuring cells dying by apoptosis.     

Synaptically-competent neurons derived from canine embryonic stem cells by lineage selection with EGF and Noggin

Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABA(A)- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model.     

Neural differentiation of human umbilical cord matrix-derived mesenchymal cells under special culture conditions

Many neural disorders are characterized by the loss of one or several types of neural cells. Human umbilical cord-derived mesenchymal cells (hUCMs) are capable of differentiating into neuron, astroglia-like and oligodendrocyte cell types. However, a reliable means of inducing the selective differentiation of hUCMs into neural cells in vitro has not yet been established. For induction of neural differentiation, hUCMs were seeded onto sterile glass slides and six various cocktails using a base medium (DMEM/LG) supplemented with 10 % FBS, retinoic acid (RA), dimethyl sulfoxide (DMSO), epidermal growth factor (EGF) and fibroblast growth factor (FGF) were used to compare their effect on neuronal, astrocyte and oligodandrocyte differentiation. The hUCMs were positive for mesenchymal markers, while they were negative for hematopoietic markers. Differentiation to adipogenic and osteogenic lineage was detected in these cells. Our data revealed that the cocktail consisting of DMEM/LG, FBS, RA, FGF, and EGF (DF/R/Fg/E group) induced hUCM cells to express the highest percentage of nestin, ß-tubulin III, neurofilament, and CNPase. The DF/Ds/Fg/E group led to the highest percentage of GFAP expression. While the expression levels of NF, GFAP, and CNPase were the lowest in the DF group. The least percentage of nestin and ß-tubulin III expression was observed in the DF/Ds group. We may conclude that FGF and EGF are important inducers for differentiation of hUCMs into neuron, astrocyte and oligodendrocyte. RA can induce hUCMs to differentiate into neuron and oligodendrocyte while for astrocyte differentiation DMSO had a pivotal role.