Human embryonic stem cells: Derivation, culture, and differentiation: A …

The ectoderm derivatives include external ectoderm, neural crest, and neural tube. These structures give rise to cells of the epidermis, external sense organs, and the peripheral and central nervous system (Gilbert, 2006).

Generating functional neurons from hESC with the aim of treating neurodegenerative diseases is the subject of intensive investigation. Shortly after derivation of the first hESC lines, Reubinoff et al. (2000) described isolation of expandable neural progenitor cells from hESC that had been cultivated for four to seven weeks at a high density in vitro. The authors illustrated that the neuroepithelium contained areas of differentiating hESC colonies, identified by expression of the embryonic polysialylated neural cell adhesion molecule (PSA-NCAM), and had distinct morphological features. These areas were mechanically dissected and expanded as neural aggregates or spheres in serum-free media. Neural induction was achieved by plating the spheres on coverslips coated with poly-D-lysine and laminin, which resulted in emergence of cells expressing the neuronal markers -tubulin and microtubule-associated protein 2 (MAP2). They also identified a subset of the neuronal cells as being glutaminergic and GABAergic neurons, as shown by the expression of glutamate and glutamic acid decarboxylase (GAD).

Reubinoff and coworkers (2001) also optimized the expansion of hESC-derived NPC by addition of B27 supplement, human recombinant epidermal factor EGF and the mitogen bFGF. The expanded NPC were capable of differentiating into all three major neural lineages (neurons, astrocytes, oligodendrocytes) in vitro as well as in vivo. Lineage tracing studies showed that NPC grafted to the ventricles of newborn mice differentiated in a region-specific manner, according to normal developmental patterning signals. For example, neuronal differentiation was specifically detected in the olfactory bulb, where postnatal neurogenesis occurs (Reubinoff et al., 2001).

At about the same time, Zhang et al. (2001) used a different protocol with similar success in generating enriched populations of NPC from hESC. In that study, differentiating EBs were treated with insulin, transferrin, progesterone, heparin, and bFGF. Continuous exposure to bFGF led to formation of monolayers of neural tube-like rosettes that were isolated by dispase enzymatic treatment. Similar to the study by Reubinoff et al. (2001), the generated NPC were capable of generating oligodendrocytes, astrocytes, and mature neurons both in vitro and after transplantation into mice.

These observations confirming the multi-lineage differentiation potential of hESC-derived NPC, and promising indications of survival and integration of these cells in vivo, set the stage for future developments of methods for selective differentiation of different neuronal phenotypes that could potentially be used to treat several central nervous system disorders. Indeed, studies examining the signals and factors that govern the proliferation and cell fate specification of neural progenitors are accumulating rapidly.

Generation of transplantable motor neurons from hESC could have potential for treating victims of spinal cord injuries, or degenerative diseases such as amyotrophic lateral sclerosis. The first functional motor neurons originating from hESC were described by Li et al. (2005). In that study, Li and colleagues used the previously described method (Zhang et al., 2001) to generate NPC cells, which were subsequently induced to become motor neurons by addition of RA to the culture medium. Further maturation to postmitotic motorneurons was induced by the ventralizing morphogenic protein SHH. An interesting aspect of this study was the specific temporal effect of RA on motor neuron induction, in that RA could induce only early, but not late neuroectodermal cells, to differentiate into motorneurons (Li et al., 2005). This occurred through upregulation of expression of HOX genes that are involved in assigning the rostrocaudal positional identity of spinal motor neurons. The functionality of the generated motor neurons was confirmed by electrophysiological experiments and establishment of neuromuscular transmission in motorneuron-myotube co-cultures (Li et al., 2005).

In another study, directed differentiation of motor neurons was achieved by addition of RA/SHH extrinsic cues to cultures of differentiating NPC generated from hESC (Lee et al., 2007). With respect to clinical potential, transplantation of hESC-derived motor neurons in developing chick embryo spinal cord showed that these cells were capable of survival and directed axonal growth over relatively long distances (Lee et al., 2007). Nevertheless, transplantation in adult rats did not result in axonal growth to outside the CNS. It should be mentioned that, although caudal and ventral patterning was achieved by RA and SHH, the initial neural induction of hESC was obtained by co-culture with MS5 mouse stromal cells, which would preclude the use of motorneurons generated by this particular strategy for any type of human transplantation therapy (Lee et al., 2007).

In a later study by Li et al. (2008), neural induction medium containing heparin and cyclic adenosine monophosphate (cAMP) in addition to RA and SHH was successful in generating a nearly homogenous population of ventral spinal progenitor cells, with highly efficient generation of motor neurons.

Promoting remyelination for treatment of neurologic disorders caused by demyelination of motor neurons is another potential application of hESC-derived cells. One of the strategies used to promote remyelination involves transplantation of oligodendrocytes, which produce the myelin sheath of motor neurons and are essential for normal signal conduction. In 2005, Keirstead and his associates demonstrated that transplantation of hESC-derived oligodendrocyte progenitor cells (OPC) produced by glial restriction media, can lead to remyelination of motorneurons, and recovery of motor function after spinal cord injury in rats (Keirstead et al., 2005). Following further assessment of the safety concerns associated with OPC transplantation in animal models (Cloutier et al., 2006), Geron Corporation obtained FDA clearance in January 2009 to begin the first human clinical trials of hESC-derived cells in the United States (see Alper 2009). OPC were generated from the H1 hESC line under current good manufacturing practices without the use of feeder cells, in defined media containing only human recombinant proteins. The Phase I trial is designed to assess the safety of transplantation of OPC in patients with acute thoracic spinal cord injuries and will be carried out at multiple medical centers.

A number of additional studies have reported the production of multiple neuronal subtypes, including cholinergic, serotonergic, GABAergic, and dopaminergic (DA) neurons, from hESC (Erceg et al., 2008; Gerrard et al., 2005; Perrier et al., 2004; Yan et al., 2005). As previously mentioned, protocols used for neural conversion of hESC generally give rise to a mixture of neuronal phenotypes. Previous studies of neural differentiation of mouse ESC have established protocols for growth factor-mediated lineage selection and survival-promoting factors of neuronal cells (Barberi et al., 2003; Lee et al., 2000; Okabe et al., 1996). As a general strategy for obtaining selective neuronal differentiation, factors with effects on the anteroposterior (AP) or the dorsoventral (DV) neuronal patterning in combination with specific neurotrophins are used at specific stages during in vitro ESC differentiation. In a comprehensive study of neural development of mouse ESC, Barberi et al. used a stromal feeder-based differentiation system to generate early ectodermal cells (6 days co-culture) and identified various combinations of factors that govern neural and neuronal subtype specification (Fig. 1) (Barberi et al., 2003). It should be noted that these differentiation strategies for mouse ESC cannot be directly applied to hESC without some modifications.

Neural subtype specification from neural progenitors derived from mouse ESC using various combinations of inducing factors. AA, ascorbic acid; bFGF, basic fibroblast growth factor; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; FGF4, fibroblast growth factor 4; FGF8, fibroblast growth factor 8; NT4, neurotrophin-4; PDGF, platelet-derived growth factor; RA, retinoic acid; SHH, sonic hedgehog. {Adapted from Barberi et al. (2003), [100]}.

To date, the majority of studies on neural differentiation of hESC have been focused on generation of dopamine producing neurons of the midbrain subtype, due to their potential application in cell replacement therapy for Parkinsons disease. Established protocols used to generate DA neurons include allowing spontaneous differentiation of hESC, followed by addition of DA inducing molecules, SHH and FGF8, and later neurotrophic factors, or by culturing hESC on feeder cells from animal or human origin that have the ability to direct hESC to become DA neurons.

Kawasaki and coworkers in Japan discovered in 2000 that certain mouse stromal cell lines had a neural and DA promoting effect on mouse ESC (Kawasaki et al., 2000). The authors showed that the activity of the stromal cells was not mimicked by FGF8/SHH, or Wnt signaling, previously known to be key factors in development and patterning of midbrain DA neurons. Thus, this strategy was established as a new approach to generate DA neurons and was termed stromal-derived inducing activity (SDIA).

Our group as well as others have adapted this approach to generate DA neurons from hESC. When the hESC line BG01 was cultured on the mouse stromal cells for three weeks, approximately 87% of colonies contained large numbers of TH+ cells (Zeng et al., 2004). The TH+ neurons generated by SDIA had midbrain characteristics, as determined by expression of Nurr1 and Pitx3 transcription factors that are strongly associated with midbrain DA neurons. The DA neurons were functional in vitro as confirmed by electrophysiological assessments and release of dopamine. However, the survival of TH+ neurons grafted into the striatum of parkinsonian rats was very limited. A parallel study of DA induction of hESC by Perrier and colleagues (2004), combined SDIA with SHH and FGF8 patterning molecules, ascorbic acid, and various neurotrophic factors including BDNF, GDNF, TGF-3, dcAMP and demonstrated that the yield and functional properties of TH+ neurons were highly dependent on exposure to SHH and FGF8.

Other feeder cells that possess DA-inducing activity, and that have been used to generate DA neurons from ESC, include testis-derived sertoli cells (Yue et al., 2006), meningeal cells (Hayashi 2008), and striatal or mesencepahlic astrocytes (Buytaert-Hoefen et al., 2004; Roy et al., 2006). Secreted factors produced by astrocytes have also been reported to promote neurogenesis and induction of DA neurons (Nakayama et al., 2003).

Yan and coworkers (2005) demonstrated neural and DA induction of hESC in the absence of any type of feeder cells by addition of SHH and FGF8 to EB-derived neural rosettes which were manually isolated from mixed cultures. The resulting TH+ neurons comprised 50%60% of the total neuronal population and were electrophysiologically active. Other differentiation paradigms have included addition of an NPC expansion step to this protocol in order to generate a more pure population of DA neurons (Cho et al., 2008).

Although mouse stromal cells that possess SDIA activity are considered as one of the most efficient tools for converting hESC to DA neurons, the use of animal cells would preclude any downstream clinical application due to possible transfer of xenogeneic material. To understand the molecular activity of SDIA, we further assessed the activity of stromal cells and found that stromal cell surface activity promoted hESC survival and was able to enhance overall neurogenesis, whereas soluble secreted factors provided DA lineage-specific instructions (Vazin et al., 2008). We then examined the gene expression profile of potent PA6 stromal cells as compared to that of cell lines lacking the DA-inducing effect (Vazin et al., 2009). Several soluble factors and growth-inducing proteins potentially responsible for the DA phenotype-promoting component of SDIA were identified, based on high levels of expression in potent DA-inducing PA6 cells. Testing of these factors showed that a combination of four factors, stromal cell-derived factor 1, pleiotrophin, insulin-like growth factor 2, and ephrin-B1, termed SPIE was sufficient to induce DA neuronal differentiation from hESC. The combination of these four factors mimicked SDIA activity, providing an approach for differentiating DA neurons from hESC in a culture system that is potentially suitable for clinical applications (Vazin et al., 2009).

Transplantation of DA precursors or neuronal cells is still at the stage where survival and integration needs to be optimized, as the majority of studies focusing on neural transplantation have reported limited or no survival of DA neurons. A few studies have, however, reported more encouraging results. A study by Roy et al. (2006) transplanted hESC-derived DA progenitors induced with immortalized human fetal midbrain astrocytes in the presence of SHH and FGF8, and illustrated that about 21% of the total number of transplanted cells (5 105 cells) were TH+. Long-lasting behavioral recovery was found in animals that received cell implants. The enhanced viability of the TH+ neurons post-transplantation may have been caused by the influence of fetal midbrain astrocytes during development or specification of these neurons.

A more recent study by Chiba and colleagues (2008) has indicated that SDIA-induced DA differentiation of hESC can be improved by addition of the BMP inhibitor noggin. Importantly, the number of TH+ cells found in animals transplanted with hESC treated with noggin was five times more (average of about 500 cells/animal) than the animals that received hESC induced by SDIA alone. The enhanced in vivo viability of TH+ cells was also reflected in animal behavioral recovery.

As previously discussed, the patterning of the neural tube along its DV and AP axis is determined by specific concentrations of morphogens including SHH, BMP, FGF and RA. Other important aspects involved in regional specification of NPC are the temporal effect of these factors, as well as the duration of signaling. There is evidence indicating that NPC progressively lose their differentiation potential and can no longer be regionally specified in response to instructive patterning cues after extended in vitro culturing (Machon et al., 2005; Santa-Olalla et al., 2003).

A recent study by Elkabetz and colleagues (2008) has identified a novel population of hESC-derived neural stem cells with a unique gene expression profile, termed neural rosette cells (R-NSC), which are isolated at an earlier stage of differentiation, as compared to the previously described NPC. Forse1 was used as a marker to isolate these early rosette stage cells, which adopted an anterior forebrain characteristic in the absence of extrinsic patterning factors. In contrast to NSC, the R-NSC could be re-specified toward caudal neuronal fates including motor neurons and midbrain DA neurons by SHH/RA and SHH/FGF8 treatment, respectively. This study also illustrated the in vivo survival and phenotype maintenance of these two rosette stage-derived neuronal phenotypes.

These findings provide evidence that neuronal plasticity of NSC is highly dependent on the developmental stage and restricted to a specific time window. Selective expansion of neural stem cells that retain their ability to differentiate towards specified neurons is of great potential value. Moreover, generation of restricted NSC has clinical relevance, as such cells have been reported to have a tendency to migrate towards the site of injury and rescue degenerating neurons following implantation in animal models (Bjugstad et al., 2008; Ourednik et al., 2002).

Characterization studies of SDIA-mediated neural induction have also suggested that midbrain regional identity can only be established during early stages of ESC differentiation (Parmar and Li, 2007). In addition, it has been suggested that early exposure of FGF8, before the onset of the neural stem cell transcription factor Sox1, is necessary for generation of DA neurons with a midbrain phenotype (Yan et al., 2005). Signaling duration is also known to affect the mechanisms that underlie the patterning role of factors. For example cells are known to respond analogously to varying concentrations of SHH, or to varying duration of exposure to this factor (Dessaud et al., 2007).

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Human embryonic stem cells: Derivation, culture, and differentiation: A ...