Use of lectins for probing differentiated human embryonic stem cells for carbohydrates

The carbohydrates present on the surface of differentiated human embryonic stem cells (hESCs) are not yet well established. Here, we have employed a panel of lectins and several anti-carbohydrate antibodies to determine the carbohydrates that are present at day 12 of hESC differentiation as embryoid bodies (EBs). On the basis of staining with fluorescein-labeled lectins, we have determined the presence of both terminal and internally linked alpha-d-mannopyranosyl groups, poly-N-acetyllactosaminyl chains, both alpha2,3- and alpha2,6-linked N-acetylneuraminic acid (Neu5Ac), alpha1,6-linked l-fucosyl, and beta-D-galactosyl groups, and more specifically, the T, Tn, and sialyl-Tn antigens. However, no alpha1,2-linked l-fucosyl, terminal nonreducing alpha-D-galactosyl, N-acetyl-beta-D-glucosaminyl, nor N-acetyl-alpha-D-galactosaminyl groups were found by this approach. We also established the presence of Neu5Acalpha2,3/2,6-Galbeta1,4 GlcNAc-terminated chains on the surfaces of 12-day-old EBs, as indicated by the great enhancement of staining by Erythrina cristagalli agglutinin (ECA) after treatment with neuraminidase. In each case, inhibition of binding by a haptenic sugar or treatment with neuraminidase was used to eliminate the possibility of nonspecific binding of the lectins. A comparison with undifferentiated cell staining revealed an increase in alpha2,3-linked Neu5Ac as well as a change to exclusively alpha1,6-linked l-fucose upon differentiation.     

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.     

Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons

Human embryonic stem (hES) cells have the ability to renew themselves and differentiate into multiple cell types upon exposure to appropriate signals. In particular, the ability of hES cells to differentiate into defined neural lineages, such as neurons, astrocytes, and oligodendrocytes, is fundamental to developing cell-based therapies for neurodegenerative disorders and studying developmental mechanisms. However, the utilization of hES cells for basic and applied research is hampered by the lack of well-defined methods to maintain their self-renewal and direct their differentiation. Recently we reported that neural precursor (NP) cells derived from mouse ES cells maintained their potential to differentiate into dopaminergic (DA) neurons after significant expansion in vitro . We hypothesized that NP cells derived from hES cells (hES-NP) could also undergo the same in vitro expansion and differentiation. To test this hypothesis, we passaged hES-NP cells and analyzed their proliferative and developmental properties. We found that hES-NP cells can proliferate approximately 380 000-fold after in vitro expansion for 12 weeks and maintain their potential to generate Tuj1⁺ neurons, GFAP⁺ astrocytes, and O4⁺ oligodendrocytes as well as tyrosine hydroxylase-positive (TH⁺) DA neurons. Furthermore, TH⁺ neurons originating from hES-NP cells expressed other midbrain DA markers, including Nurr1, Pitx3, Engrail-1, and aromatic l -amino acid decarboxylase, and released significant amounts of DA. In addition, hES-NP cells maintained their developmental potential through long-term storage (over 2 years) in liquid nitrogen and multiple freeze–thaw cycles. These results demonstrate that hES-NP cells have the ability to provide an expandable and unlimited human cell source that can develop into specific neuronal and glial subtypes.     

利用胚胎干细胞分化的拟胚体研究小鼠早期胚胎发育过程

小鼠胚胎干细胞(ES细胞)具有分化的全能性已经得到广泛共识。ES细胞在体外分化所形成的拟胚体在结构上能够模仿早期胚胎发育过程,包括在内细胞团表面形成内胚层、柱状上皮细胞的分化,以及中央空腔的形成。本文介绍利用拟胚体研究小鼠早期胚胎发育过程中各个胚胎阶段的发育、细胞程序性死亡的发生及TGF-β信号在胚胎发育过程中的作用。     

A simplified method to generate serotonergic neurons from mouse embryonic stem and induced pluripotent stem cells

We have developed a new simple method to induce serotonergic neurons from embryonic stem (ES) and induced pluripotent stem cells. When ES or induced pluripotent stem cells were cultured on a thick gel layer of Matrigel, most colonies extended TuJ1-positive neurites. We found that noggin, a known antagonist of bone morphogenic protein, induces ES cells to express genes involved in serotonergic differentiation, such as Nkx2.2, Pet-1, Sonic hedgehog, tryptophan hydroxylase 2, and serotonin transporter, as well as increases high potassium-induced release of serotonin. To concentrate serotonergic neurons, ES cells carrying Pet-1-enhancer-driven enhanced green fluorescent protein were differentiated and sorted into about 80% pure cultures of serotonergic neurons. Whole cell voltage-clamp recordings showed a voltage-dependent current in dissociated neurons. This simplified method provides an alternative option for serotonergic differentiation of pluripotent stem cells and will likely contribute a deeper understanding regarding the nature of serotonergic neurons and open new therapeutic perspectives for the treatment of psychiatric disorders.     

Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation

Embryonic stem cells (ESCs) are pluripotent cells capable of differentiating into all somatic and germ cell types. The intrinsic ability of pluripotent cells to generate a vast array of different cells makes ESCs a robust resource for a variety of cell transplantation and tissue engineering applications, however, efficient and controlled means of directing ESC differentiation is essential for the development of regenerative therapies. ESCs are commonly differentiated in vitro by spontaneously self‐assembling in suspension culture into 3D cell aggregates called embryoid bodies (EBs), which mimic many of the hallmarks of early embryonic development, yet the 3D organization and structure of EBs also presents unique challenges to effectively direct the differentiation of the cells. ESC differentiation is strongly influenced by physical and chemical signals comprising the local extracellular microenvironment, thus current methods to engineer EB differentiation have focused primarily on spatially controlling EB size, adding soluble factors to the media, or culturing EBs on or within natural or synthetic extracellular matrices. Although most such strategies aim to influence differentiation from the exterior of EBs, engineering the microenvironment directly within EBs enables new opportunities to efficiently direct the fate of the cells by locally controlling the presentation of morphogenic cues. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures

Derivation of human neural progenitors (hNP) from human embryonic stem (hES) cells in culture has been reported with the use of feeder cells or conditioned media. This introduces undefined components into the system, limiting the ability to precisely investigate the requirement for factors that control the process. Also, the use of feeder cells of non-human origin introduces the potential for zoonotic transmission, limiting its clinical usefulness. Here we report a feeder-free system to produce hNP from hES cells and test the effects of various media components involved in the process. Five protocols using defined media components were compared for efficiency of hNP generation. Based on this analysis, we discuss the role of basic fibroblast growth factor (FGF2), N2 supplement, non-essential amino acids (NEAA), and knock-out serum replacement (KSR) on the process of hNP generation. All protocols led to down-regulation of Oct4/POU5F1 expression (from 90.5% to <3%), and up-regulation of neural progenitor markers to varying degrees. Media with N2 but not KSR and NEAA produced cultures with significantly higher (p<0.05) expression of the neural progenitor marker Musashi 1 (MSI1). Approximately 89% of these cells were Nestin (NES)+ after 3 weeks, but they did not proliferate. In contrast, differentiation media supplemented with KSR and NEAA produced fewer NES+ (75%) cells, but these cells were proliferative, and by five passages the culture consisted of >97% NES+ cells. This suggests that KSR and NEAA supplements did not enhance early differentiation but did promote proliferating of hNP cell cultures. This resulted in an efficient, robust, repeatable differentiation system suitable for generating large populations of hNP cells. This will facilitate further study of molecular and biochemical mechanisms in early human neural differentiation and potentially produce uniform neuronal cells for therapeutic uses without concern of zoonotic transmission from feeder layers.     

Peripheral sensory neurons differentiate from neural precursors derived from human embryonic stem cells

Abstract Neural precursors have been derived from human embryonic stem cells (hESC) using the bone morphogenetic protein antagonist noggin. These neural precursors can be further differentiated to produce neural cells that express central nervous system (CNS) markers. We have recently shown that naïve hESC can be directed to differentiate into peripheral sensory (PS) neuron-like cells and putative neural crest precursors by co-culturing with PA6 stromal cells. In the present study, we examine whether hESC-derived neural precursors (NPC) can differentiate into the peripheral nervous system, as well as CNS cells. As little as 1 week after co-culture with PA6 cells, cells with the molecular characteristics of PS neurons and neural crest are observed in the cultures. With increased time in culture, more PS-like neurons appear, in parallel with a reduction in the neural crest-like cells. These results provide the first evidence that neural precursors derived from hESC have the potential to develop into PS neurons-like as well as CNS-like neuronal cells. About 10% of the cells in NPC-PA6 co-cultures express PS neuron markers after 3 weeks, compared with <1% of hESC cultured on PA6. This enrichment for peripheral neurons makes this an attractive system for generation of peripheral neurons for pathophysiology study and drug development for diseases of the peripheral nervous system such as Familial Dysautonomia and varicella virus infection.     

Derivation of neural precursor cells from human ES cells at 3% O(2) is efficient, enhances survival and presents no barrier to regional specification and functional differentiation

In vitro stem cell systems traditionally employ oxygen levels that are far removed from the in vivo situation. This study investigates whether an ambient environment containing a physiological oxygen level of 3% (normoxia) enables the generation of neural precursor cells (NPCs) from human embryonic stem cells (hESCs) and whether the resultant NPCs can undergo regional specification and functional maturation. We report robust and efficient neural conversion at 3% O(2), demonstration of tri-lineage potential of resultant NPCs and the subsequent electrophysiological maturation of neurons. We also show that NPCs derived under 3% O(2) can be differentiated long term in the absence of neurotrophins and can be readily specified into both spinal motor neurons and midbrain dopaminergic neurons. Finally, modelling the oxygen stress that occurs during transplantation, we demonstrate that in vitro transfer of NPCs from a 20 to 3% O(2) environment results in significant cell death, while maintenance in 3% O(2) is protective. Together these findings support 3% O(2) as a physiologically relevant system to study stem cell-derived neuronal differentiation and function as well as to model neuronal injury.     

Genetic approach to track neural cell fate decisions using human embryonic stem cells

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry⁺NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.     

Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation

The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy and tissue engineering depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe for the first time the formation of differentiating human embryoid bodies (hEBs) in rotating bioreactors to try and control their agglomeration. The efficacy of the dynamic process compared to static cultivation in Petri dishes was analyzed with respect to the yield of hEB formation and differentiation. Quantitative analyses of hEBs, DNA and protein contents, and viable cell concentration, as measures for culture cellularity and scale-up, revealed 3-fold enhancement in generation of hEBs compared to the static culture. Other metabolic indices such as glucose consumption, lactic acid production, and pH pointed to efficient cell expansion and differentiation in the dynamic cultures. The type of rotating vessel had a significant impact on the process of hEB formation and agglomeration. In the slow turning lateral vessel (STLV), hEBs were smaller in size and no large necrotic centers were seen, even after 1-month cultivation. In the high aspect rotating vessel (HARV), hEB agglomeration was massive. The appearance of representative tissues derived from the three germ layers as well as primitive neuronal tube organization, blood vessel formation, and specific-endocrine secretion indicated that the initial developmental events are not altered in the dynamically formed hEBs. Collectively, our study defines the culture conditions in which control over the aggregation of differentiating hESCs is obtained, thus enabling scaleable cell production for clinical and industrial applications.     

Embryonic stem cell-derived neural progenitors as non-tumorigenic source for dopaminergic neurons

AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.     

Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.