Critical steps for rapid and efficient neuralization of ESCs
For derivation of neural precursors, ESC cultures () were collected by collagenase-IV treatment 5 days after plating. Cell clusters were allowed to settle, and the supernatant containing floating cells was discarded. The ESC clusters were triturated to dislodge loosely attached cells and reduce cluster size to 50-100 cells. Reducing the cluster size is critical for efficient neuralization because large aggregates tend to develop cavities resulting in increased cell death and generation of nonneural lineages. ESC clusters were transferred to a suspension medium (SM) and allowed to grow as spheres for 6 days, with a change in medium on alternate days (). The spheres were collected, gently triturated and plated on polyornithine-coated plates in expansion medium. The cultures appeared to be entirely composed of columnar neuroepithelial rosettes, with bipolar/triangular cells radiating outward. The remaining floating clusters developed multiple rosettes as well (). After 4-6 days in expansion medium, the C-NPCs were replated in the same medium, forming monolayer cultures of C-NPCs (). The cultures could be robustly amplified for up to five passages, after which most cells would stop proliferating and spontaneously differentiate. Both H9 and H14 lines yielded similar results. Data from the H9 cell line are presented below.
Figure 1 Schematic representation of C-NPC derivation process. (a) Stage 1: colonies of undifferentiated human ESCs (H9 line). Pristine ESC cultures are critical for this protocol. To monitor the quality of ESC culture conditions, we used a separate ESC line engineered (more ...)
Reproducible proteomic signature of C-NPCs
To investigate the reproducibility and the robustness of rapid neuralization, we performed a comparative whole-cell proteomic analysis of two independent C-NPC derivations: (1) C-NPCs derived from ESCs at passage 35 and (2) C-NPCs derived from the same culture of ESCs that were propagated for an additional 55 passages (corresponds to an amplification of approximately 1015
-fold, if the entire culture were to be expanded). Control cells included adult brain-derived neural precursors (kind gift of Dr. Phil Schwartz, Children's Hospital of Orange County, Ca, USA) grown for several weeks in the same medium as C-NPCs, and the human acute monocytic leukemia cell line THP-1. We employed SELDI-MS technology (Ciphergen Biosystems Inc., Fremont, CA, USA), which combines selective adsorption onto a pretreated chip with linear TOF mass spectrometry. This technology allows fast, cost-effective, and comprehensive comparison of proteomic profiles or signatures from multiple samples. We found that the profiles of protein expression were nearly identical for the two C-NPC cultures derived many months apart (). Despite the overall similarity between the brain-derived NPCs and C-NPC profiles, differences in the relative peak sizes can be readily discerned. The signature of the human acute monocytic leukemia THP-1 cells showed major differences from both NPC preparations. This analysis demonstrates the robust and reproducible generation of C-NPC cultures after long-term propagation (large-scale expansion) of ESCs. Although C-NPCs derived by rapid neuralization cannot be amplified beyond five passages, the straightforward and reproducible differentiation offers an easy scale-up strategy by generating a large number of C-NPCs directly from ESCs. These C-NPCs have been shown to retain the correct diploid karyotype after long-term propagation (up to 100 passages) using appropriate culture conditions.20,21
Figure 2 Proteomic profiling of C-NPCs and fetal brain-NPCs. Surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS), whole-cell proteomic profiling of C-NPCs. Left panel represents the cellular proteomic profile between 30-60 and 75-150 kDa. (more ...)
In situ characterization of C-NPCs
First, using immunochemistry and RT-PCR, we focused on known markers of neural precursors and undifferentiated cells in C-NPC cultures (). We found the C-NPC cultures (after 10 days of differentiation) stain uniformly positive for Sox2, Musashi1, and Nestin, and negative for Oct4, Nanog, MAP2, and GFAP (). TuJ1-positive young neurons were extremely rare, confirming the undifferentiated nature of the C-NPC cultures (). RT-PCR analysis confirmed the absence of transcripts for Oct4 and Nanog, pluripotent ESC markers, GATA-1, a marker of primitive and definitive hematopoiesis, GATA-4, a marker for pharyngeal endoderm and cardiac derivatives, Nkx2.5, a marker of cardiac mesoderm, and PDX-1, a pancreatic tissue marker. Thus, after 10-12 days of differentiation, these cultures were uniformly positive for the neuroectodermal markers and uniformly negative for mesodermal, endodermal, and mature neuronal and glial markers (). These results suggest that the majority of ESCs differentiated into neuroectoderm under these defined conditions, although some nonneural lineages might have been generated and subsequently died off.
Figure 3 C-NPCs express a homogeneous array of proneural markers. (a) Analysis of markers characteristic for undifferentiated ESCs, mesoderm, and endoderm using RT-PCR in human ESCs and C-NPCs (day 12 of differentiation). (b) Immunostaining for developmental markers; (more ...)
Freshly generated C-NPCs were uniformly negative for GFAP (); however, on passaging, cells initially emigrating from clusters and, eventually, all cells in the culture stained positively for GFAP (). This expression pattern clearly differs from brain-derived human NPCs, which are uniformly GFAP-positive, even during early passages.22
Previous work suggested that these cells represented the radial glial phenotype of mouse ESC-derived NPCs.16,23
The acquisition of GFAP staining and the morphology of the C-NPCs at this stage are consistent with a radial glia identity.
To further address the identity of the C-NPCs, we used microarray technology to examine the mRNA expression of CNS markers of regional specification during ESC neuralization. Both anterior (Emx2, Otx2, Dlx1/2) and posterior (HoxA2, HoxB2, HoxB6) CNS markers were detected ( and ). Some of the markers, such as Pax2, Gbx2, and HoxB6, were transiently upregulated during ESC neuralization to C-NPCs. In contrast, Otx2 was found to be expressed in ESCs but was downregulated during neuralization (). mRNA for several other genes (e.g., FoxG1, Emx1, Nkx2.1, En1, Nkx2.6, HoxA1, HoxC5) was not detected using microarray, either due to lack of expression or low hybridization efficiency of the probes. To corroborate these microarray data, immunostaining for the anterior marker Otx2 and the posterior marker HoxB4 was performed after 7 days (168 h) of neuralization (). We found that the majority of day 7 C-NPCs were positive for both Otx2 and HoxB4 but with variable levels of expression. These results were obtained using medium containing B27 supplement with or without retinoic acid. The expression of mRNAs for dorsal-ventral spinal cord markers, such as Math1, Ngn1, Nkx6.1, and Nkx2.1, was not detected using microarray analysis (data not shown). These results suggest that C-NPCs are not regionally specified under our conditions and likely have broad developmental potential.
Figure 4 C-NPCs express both anterior and posterior markers of neuroectoderm. (a) Microarray analysis of anterior-posterior markers during the first 10 days of neuralization. Only mRNA transcripts with at least twofold changes are shown. Upregulated transcripts (more ...)
Figure 6 Gene expression analysis of hESC neuralization. (a) Volcano statistical analysis (GeneSpring) revealed about 300 genes that changed significantly (>2-fold, P>0.05) between the undifferentiated ESC state (time 0) and C-NPC stage (240 h (more ...)
Efficient neuralization of ESCs does not require exogenous BMP inhibitors
The BMP inhibitor Noggin, commonly used to enhance neural cell fate, was not included in these defined conditions. To determine whether Noggin is dispensable for efficient neuralization of ESCs, we tested three conditions in parallel: (1) our original defined conditions; (2) original defined conditions + 100 ng/ml Noggin added during the first 5 days of differentiation; (3) original defined conditions + 20 ng/ml of BMP2 and 20 ng/ml of BMP4 added during the first 5 days of differentiation. If exogenous Noggin is dispensable for ESC neuralization, then the addition of Noggin to our defined conditions should not change the outcome of differentiation. In contrast, the addition of BMP2/4 should activate the TGFβ pathway, resulting in an altered gene expression profile and the appearance of nonneural cell fates, similar to those seen in embryoid bodies. To test this hypothesis, we monitored global gene expression changes during differentiation and investigated the status of BMP/TGFβ signaling (). Constant levels of Smad1 and Smad4 mRNA, but not Noggin or BMP4, were detected during ESC neuralization under our neuralization conditions. BMP5 expression was upregulated at later time points concomitant with the downregulation of the BMP-inhibitor follistatin (). We did not observe statistically significant alterations in gene expression (averaged over the entire differentiation period) on addition of Noggin.
Figure 5 Exogenous BMP-inhibitors (such as Noggin) are not required for efficient neuralization of hESCs. (a) Gene expression dynamics of the Smad signaling pathway (lllumina microarrays). (b) Normalized gene expression (averaged over days 3-13 of differentiation) (more ...)
In contrast, BMP-treated cultures revealed major changes in gene expression (). Characteristically, BMP treatment resulted in downregulation of proneural genes such as Sox3 and Pax6, and upregulation of mesodermal markers such as simple epithelial keratins 8/18/19 and lymphoid or colon epithelium-associated Hox genes (B8, C6, B6). In contrast, Noggin treatment did not result in differential expression of these genes (). Consistent with the microarray results, phosphorylation of Smad1/5 was barely detectable in the original cultures as well as in Noggin-treated cultures. As expected, robust phosphorylation of Smad1/5 was detected in BMP-treated cultures (). Thus, during our protocol for rapid neuralization of ESCs, we observed very low BMP/TGFβ signaling. Taken together, these results provide strong evidence that exogenous Noggin is dispensable for rapid and uniform neuralization of human ESCs.
Two major waves of molecular expression during ESC neuralization
To gain insight into the global molecular changes occurring during C-NPC derivation, we performed a functional whole-genome analysis, which provides a very detailed, unbiased, and robust characterization of the state of the cells. Genome analysis shows the stages that cells go through, validating their identities by identifying known markers of cell type or state, and providing a formal, unbiased basis for molecular characterization of neural progenitor differentiation.
We collected expression data at 15 time points over 10 days of differentiation, spanning the stages of undifferentiated ESCs, rosette formation, and appearance of lattice-like monolayers. We focused on the genes that changed significantly between time 0 (undifferentiated ESCs) and a given time point. At day 10 of differentiation, situated at the transition between the ESC and C-NPC stages, more than 300 mRNA transcripts were found to be up- or downregulated with various kinetics (). Quantification of the kinetics of expression revealed two waves of global gene expression change: the first wave occurred at 72 h and the second at 144 h of differentiation (; Supplementary Figure 1A
). Remarkably, the first wave of change occurred in the absence of major morphological alterations and preceded the formation of columnar neuroepithelial rosettes. In particular, transcripts of FGF5, a marker of primitive ectoderm in the mouse,24
were transiently upregulated threefold at 72 h (first wave) but was subsequently downregulated to the basal level found in ESCs by 144 h (Q-PCR; Supplementary Figure 1B
). To define the pathways and developmental stages that can be associated with the gene expression changes detected in the first and second waves, we performed meta-analysis of all publicly available microarray data. We employed the NextBio search engine (www.nextbio.com
) that allows queries of the existing datasets using a particular gene set, including the fold change for each gene in the set. Comparison with available datasets for mouse ESC neuralization25
revealed many genes that were downregulated in both the first wave of human ESC neuralization and in mouse (comprising ~60% of all gene changes in the first wave). During the second wave, a significant number of both up- and downregulated genes were shared with mouse neural progenitors (representing ~80% of all gene changes in the second wave), suggesting that the second wave of changes in gene expression is likely associated with neural commitment (Supplementary Figure 2A and C
Indeed, querying developmental expression datasets revealed that the gene expression profile in the first wave was associated with early developmental events such as organ development, regionalization, organ morphogenesis, as well as neural crest and neural tube development. In contrast, the second wave was more selectively associated with later events such as central nervous system development, brain development, and neurogenesis (Supplementary Figure 2B and C
; see Supplementary Tables 1 and 2
for a complete list of gene changes during the first and second waves, respectively). The percentage of differentially regulated transcripts that varied transiently dropped from 80 to 20% between 48 to 144 h of differentiation and then dropped further below 5% at 240 h (). Taken together, these results suggest the presence of a transient state characterized by differential gene expression between 48 and 144 h of differentiation, which precedes the formation of neuroepithelial rosettes. On the other hand, stable gene expression profiles, likely corresponding to a defined cell population, emerge at approximately 10 days of differentiation.
Similar to other reports on human ESC neuralization, we observed downregulation of Oct4, Nanog, UTF1, E-cadherin, and Lefty, consistent with complete loss of the pluripotent phenotype typical of human ESCs. Concomitantly, we observed upregulation of FABP7, PAX6, Dlx, MEF2C, and Sox9, consistent with acquisition of the neural phenotype ().26,27
However, we observed faster downregulation of known pluripotency markers, such as Oct4 and Nanog, compared to previously published neuralization procedures.13-15
We also found that neural-related transcripts were induced with different kinetics, consistent with their role in neural development. For example, the radial glia marker, FABP7, and the transcription factor regulating gliogenesis and development of neural crest lineage, Sox9, were upregulated very early during ESC differentiation (in the first wave of gene expression change), corresponding to the their developmentally controlled temporal expression.28
In contrast, transcription factors such as Dlx1/2 and MEF2C, which are involved in neuronal specification and migration, were upregulated during the second wave of differentiation, again consistent with their expression during neuronal developmental ().29
In addition to the selective survey of known genes, we used an unbiased approach, based on unsupervised hierarchical clustering (GeneSpring software). This analysis grouped the samples into four main clusters, which correspond to discrete temporal epochs during ESC neuralization (). The first cluster consisted of undifferentiated ESCs, encompassing the genes associated with the pluripotent state. The second cluster encompassed the `latent period' between 12 and 48 h of differentiation, which was characterized by minimal changes in gene expression when compared to ESCs. Only a few genes (25-30) within the second cluster varied at any time point. This cluster consisted primarily of genes whose up- or downregulation was transient, and most genes (70-80%) returned to baseline after this period. The third and the fourth clusters corresponded to the two major waves of change in gene expression (days 3 and 6), as identified above through differential gene expression analysis, thus providing an independent validation for these findings.
Curiously, several developmentally controlled genes were transiently upregulated, peaking at approximately 120 h, a time in between the two major waves of change in gene expression. These genes include: LMO1, IRX1, and PAX2. LMO1 is a transcriptional regulator involved in the Hox-dependent regulatory network for hindbrain patterning and contributes to leukemogenesis in concert with OLIG2; IRX1 is a member of the Iroquois homeobox gene family, playing multiple roles during pattern formation of vertebrate embryos; and PAX2 is a paired box transcription factor, implicated in the renal-coloboma syndrome characterized by ocular, renal, and CNS anomalies. Although the roles of LMO1, IRX, and PAX2 in neurogenesis have not been documented because of the timing of their expression, we speculate that these genes may play critical roles in neuralization of ESCs, in particular during very early specification of neuroepithelial fates.
Analysis of gene coregulation identifies several functional modules
To move beyond lists of differential expression and explore the higher-order organization of gene expression at a systems level as it relates to ESC neuralization, we applied a weighted gene coexpression network analysis (WGCNA).30
Viewing the dataset in this manner provided a complementary framework for analysis of transcriptional regulation during ESC neuralization. We and others have shown that this method allows one to define the transcriptome structure and to visualize modules of coexpressed genes that correspond to functional units.30,31
Briefly, the absolute Pearson correlations between any one gene and every other gene on the array were computed, weighted, and used to determine the topological overlap (TO), which is a measure of connection strength or neighborhood sharing. Then, genes were clustered based on TO, and groups of highly interconnected genes (modules, identified by color names) were identified (). Modules were annotated using gene ontology (GO) and ingenuity pathway analysis (www.ingenuity.com
) to aid in assigning the functional relevance of genes and pathways in each module (). WCGNA identified two major types of modules present during ESC neuralization. The first type was characterized by a group of genes that coordinately change expression levels around 3-5 days of differentiation (). Within this class, we found modules characterized by changes occurring early (day 3: DeepSkyBlue module) and late (day 5: Pink module), consistent with the two waves of expression changes identified by the standard analysis for differential expression (). Several modules (Gold, Honeydew, and Green) showed pronounced biphasic behavior, following the transition points at days 3 and 5 but with minimal changes in between (). This result is consistent with the emergence of the transient but defined genetic/cellular state during ESC neuralization as proposed above. The WGCNA analysis also revealed upregulation of several classical pathways, such as Wnt/β-catenin, integrin, leukocyte extravasation signaling, G2
/M checkpoint regulation, and nicotinamide metabolism, known to play roles in ESC neuralization and to be functionally important in neural precursors. Indeed, the role of the Wnt/β-catenin pathway in the neuralization process,32
the critical importance of leukocyte extravasation signaling in neural stem cell-mediated immunomodulation,33
and the intriguing role of metabolism in proliferation of neural precursors34
have been previously documented. Together, these findings validate the WGCNA analysis performed here.
Functional GOTERM annotation of the expression modules (represented by the Hub genes) identified by WGCNA/Ingenuity analysis of hESC differentiation into neural progenitors
Functional annotation showed that the pathways represented in the early DeepSkyBlue module are involved in the regulation of programmed cell death, the IκB/NF-κB pathway, and morphogenesis (). The early Green module encompasses genes involved in development of the nervous system and neuronal differentiation, including cytoskeletal proteins, small GTPases, and transcriptional regulators. The late Gold module represents genes involved in cellular metabolism and intracellular protein transport. The late Pink module includes genes involved in the control of the M phase of the cell cycle, regulators of RNA Polymerase II, proteins with GTPase activity, and zinc-finger containing proteins. This analysis identified novel candidate pathways and genes likely to play roles in ESC neuralization (see description of the `Hub' genes below).
The second major class of modules showed changes at very early time points. Characteristically, these changes were transient, mostly reverting to baseline after day 5 of neuralization (). In this case, WGCNA complemented the standard analysis, demonstrating that the changes occurring at early time points were not noise but rather exemplary of functional organization. These results are consistent with the previous analysis suggesting the presence of a transient genetic/cellular state between 72 and 120 h and the emergence of a more definitive cell population after 144 h of neuralization. Functional annotation of these modules revealed involvement of zinc-finger transcriptional regulators (DarkBlue),RNA metabolism and ribosome assembly (Salmon), and glucose and carbohydrate metabolism (Rosy-Brown). Collectively, in these modules, several canonical pathways, affecting estrogen, glutamate, death receptor signaling, peroxisome proliferator-activated receptor (PPAR) signaling, and oxidative phosphorylation, were found to be transiently upregulated. Glycolysis/gluconeogenesis, pentose phosphate, fructose, mannose, starch, and sucrose metabolism were transiently downregulated (RosyBrown). This finding is consistent with a connection between intermediary energy metabolism and progenitor cell proliferation.34
Thus, in addition to known genes and signaling cascades, a number of new pathways were identified by the WGCNA approach, providing the impetus for future investigations into their potential role in neural development.
Another advantage of WCGNA is that each functional module is represented by a collection of Hub genes, which are defined as the `most connected' or most central genes within the module's network. The expression dynamic of a Hub gene represents the first principal component of the expression profiles within a module.30
We have identified a number of Hub genes within each module, and their expression profile and connectivity with other genes suggest intimate involvement in the neuralization process (; Visant or spider plots, Supplementary Figures 3-11
). This approach also provides the basis for gene discovery, using a `guilt by association' approach.30,35
Indeed, our analysis has identified many hypothetical Hub genes, suggesting their importance for future functional studies. For example, the Green module encompasses genes and pathways classically associated with neuronal differentiation and CNS development ( and ). The graphical representation of gene connectivity in the Green module (Supplementary Figure 8
) illustrates that several of the most connected genes (Hubs) are well-known key regulators of neuronal differentiation and CNS development. As corroboration, RAI1 deficiency in mice causes learning impairment and motor dysfunction,36
and JAM3-deficient mice exhibit loss of integrity of the myelin sheath and defective nerve conduction.37
Hub genes such as TMSNB, MLLT3, and MAGED2 were previously associated with various forms of neoplasia and are highly connected to RAI1. We propose that these and other Hub genes identified in this study are candidates as key players in ESC differentiation into neural stem/progenitor cells, not only in vitro
but also in vivo
during normal human development.
The Hub genes identified by the WGCNA analysis of hESCs differentiation into neural progenitors
In vitro differentiation of C-NPCs
With our approach, we were able to efficiently differentiate cultures of homogeneous C-NPCs into the three major neural subtypes: neurons, astrocytes, and oligodendrocytes (). Abundant neurons, staining positively for βIII-tubulin (TuJ1), MAP2, and NSE were detected after 2-3 weeks of differentiation (). Bipolar as well as multipolar stellate GFAP-positive astrocytes were also detected (; Supplementary Figure 11
). In mice, dividing GFAP-expressing precursors in the adult subependymal zone have a bipolar or unipolar phenotype that differs from nondividing multipolar stellate astrocytes.38
In the absence of a robust marker that distinguishes multipotent dividing neural precursor cells from mature astrocytes, the distinctive cell morphologies of GFAP-positive cells suggest (but, certainly, do not prove) the presence of both cell types during our in vitro
differentiation of C-NPCs. Surprisingly, after 2 weeks of differentiation, many O1-positive cells were observed, suggestive of oligodendrocytes (). Costaining for O1 and TuJ1 revealed numerous O1-positive processes that closely paralleled the TuJ1-positive neuronal processes (). After 3 weeks of differentiation, multiple processes that stained positively for MBP were also detected (). Many processes also stained for both neuron-specific enolase (NSE) and MBP (). The close juxtaposition of neuronal (TuJ1) and oligodendrocyte (O1, MBP) markers raises the possibility that some C-NPCs differentiated into oligodendrocytes, which in turn initiated in vitro
myelination of neuronal processes generated in the same culture.
Figure 7 Differentiation of C-NPCs results in abundant neurons and oligodendrocytes capable of in vitro myelination of neuronal processes. (a-c) Young neurons stained for βIII-tubulin (TuJ1). Cell nuclei are counterstained with DAPI. (b-f) Various types (more ...)
Electrophysiology and digital calcium imaging of neurons derived from C-NPCs
To address the functional properties of neurons detected by immunostaining, we performed calcium imaging and patch-clamp recording. C-NPC cultures that had been differentiated for 4 weeks were loaded with Fura-2/AM and exposed to N-methyl-D-aspartate (NMDA). We observed robust Ca2 + responses in individual cells from several independent cultures (), suggesting the presence of the NMDA-subtype of glutamate receptor on at least 0% of cells in the cultures.
Figure 8 Characterization of differentiated C-NPCs as neurons using physiological criteria. NMDA evoked increases in [Ca2+] in numerous cells derived from C-NPCs that had been cultured for 2-4 weeks. (a) Fura-2-loaded cells were exposed to NMDA (100 μM (more ...)
To unambiguously document the presence of these receptors as well as other ligand-and voltage-gated currents that should be develop in maturing neurons, we performed whole-cell recordings with patch electrodes on C-NPCs after 4 weeks of differentiation. About 10% of the cells displayed action potentials typical of neurons (). The sodium currents underlying these action potentials were blocked by 0.1 mM tetrodotoxin (TTX) and manifested a fast time course (). Potassium and calcium channels were also detected (data not shown). After 3 weeks in culture, approximately 30% of the cells exhibited glutamate-evoked currents that could be partially blocked by D-APV, a specific antagonist of NMDA-type glutamate receptors (). In four cells, we observed robust γ-aminobutyric acid (GABA)-evoked currents, indicating that these cells could respond to this inhibitory transmitter (). Miniature excitatory postsynaptic currents (mEPSCs), which reflect functional synaptic activity, were detected after 3 weeks of differentiation (). Taken together, these findings suggest that a proportion of the C-NPCs differentiated sufficiently to display a variety of electrophysiological properties typical of bona fide neurons.
C-NPCs survive, migrate, and differentiate on transplantation into the neonatal mouse brain
Human ESC-derived NPCs were transduced with a PGK-eGFP lentiviral cassette and transplanted into the lateral ventricles of newborn CD1 mice (~105 cells per animal). Ten weeks later, the animals were sacrificed and brain sections analyzed for the presence of cells expressing the eGFP marker. Many human eGFP-labeled cells were detected in various regions of the brain, including cells with morphologies characteristic of newly generated olfactory bulb granule neurons (). Cells with complex, branched morphologies and recognizable dendritic spines (characteristic of neuronal cells) were detected in the cortex (). Rare cells with round cell bodies and no processes were found in the SVZ and choroid plexus (). Cell bodies in the cortex were often found in clusters (), probably due to additional rounds of division after transplantation. These results demonstrate that on transplantation into the mouse brain, C-NPCs can survive, migrate, and differentiate into cells with morphologies typical of neuronal cells types. To unequivocally prove the neuronal identity of these differentiated C-NPCs, we used immunocytochemistry with specific neuronal markers viewed under confocal microscopy (). At 12 weeks after transplantation, many eGFP-expressing cells were found to be positive for the neuronal marker MAP2, consistent with the notion that these human C-NPCs had differentiated into neurons after transplantation ().
Figure 9 hESC-NPC transplantation into the neonatal mouse brain. hESC-NPCs engineered with a PhG-eGFP cassette were transplanted into the lateral ventricle of newborn CD1 mice (n =6) and analyzed 10 weeks later. Upper left: camera lucida shows the position of (more ...)
Figure 10 Immunostaining of mouse cortex following transplantation of C-NPCs. C-NPCs engineered with a PGK-eGFP cassette were transplanted into the lateral ventricle of newborn CD1 mice and analyzed after 12 weeks. (a-c) Colocalization of eGFP-positive, transplanted (more ...)
C-NPCs do not hyperproliferate or form tumors in vivo
Previous studies had shown that ESCs transplanted into SCID mice resulted in teratoma formation,8,39,40
and even transplantation of ESC-derived neural stem cells had resulted in neural cell hyperproliferation or frank malignancy.1,41
In contrast, in this study, none of the newborn mice injected with C-NPCs (ranging from 105
cells per animal) developed discernable brain tumors or neoplasia on examination of brain sections at 2-15 weeks after transplantation (20 mice analyzed over a 1-year period). Similarly, none of six adult mice injected intravenously with C-NPCs developed pathological overgrowth. Additionally, no teratomas were observed after intramuscular injection of 3×106
C-NPCs into SCID mice; injection of 1×106
ESCs produced teratomas as expected (data not shown). These results are consistent with complete loss of pluripotent phenotype in C-NPCs, which stain negatively for Oct4 and Nanog, and limited capacity for in vivo proliferation.