in vitro differentiation of human parthenogenetic stem...

37ISSN 1746-075110.2217/RME.11.110 © 2012 Future Medicine Ltd Regen. Med. (2012) 7(1), 37–45

Research Article Research Article

In vitro differentiation of human parthenogenetic stem cells into neural lineages

Unfertilized human oocytes can be artificially activated by appropriate chemical stimuli to develop into parthenogenetic blastocysts. The inner cell mass of such blastocysts can be iso-lated and expanded as stable stem cell lines. First intentionally obtained by Revazova et al., human parthenogenetic stem cells (hpSCs) are similar to biparental human embryonic stem cells (hESCs) in their proliferation capacity and multilineage in vitro differentiation [1,2].

hpSCs can be either heterozygous or homo-zygous depending on the way the genome forms from the maternal chromosome set. Homozygous hpSCs may be useful as a source of cells for transplantations since the set of HLA genes in hpSC is able to produce differ-entiated derivatives less susceptible to immune rejection. Furthermore, if the HLA type is common, differentiated derivatives will match many millions of individuals [1,3]. In addition to these immunogenetic advantages, as parthe-nogenesis does not involve the destruction of a viable human embryo, the use of hpSCs does not raise the same ethical concerns as conventional hESCs. Thus, hpSCs are an attractive alterna-tive to other pluripotent stem cells as a source of somatic cell lines, including the multipotent neural stem cells (NSCs).

NSCs are self-renewing multipotent stem cells of the nervous system, which have the capacity to differentiate into neurons, oligodendrocytes and astrocytes [4]. NSCs can be obtained directly from a fetus and the adult CNS or by means of induced neural differentiation from pluripotent stem cells. NSCs are able to proliferate in vitro without losing their differentiation capacity for a relatively long time, and hence provide reserves of cell material for further applications. NSCs are considered to be a prospective therapy for neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s dis-ease and so on, as well as for immobility caused by spinal cord injuries. Successful experiments using animal models confirm efficiency of NSC therapy [5].

The capacity to differentiate into neurons and glial cells was experimentally proved for mice [6], primates [7], and hpSCs [1,2,8]. Parthenogenetic stem cells bear two sets of maternally and no paternally imprinted genes, which is assumed to represent an obstacle for the differentiation into derivatives of all three germ layers. Experiments with chimeric animals reveal uneven selective processes, operating against parthenogenetic cells within certain differentiation pathways during fetal and postnatal development; at the

Human parthenogenetic stem cells are derived from the inner cell mass of blastocysts obtained from unfertilized oocytes that have been stimulated to develop without any participation of male gamete. As parthenogenesis does not involve the destruction of a viable human embryo, the derivation and use of human parthenogenetic stem cells does not raise the same ethical concerns as conventional embryonic stem cells. Human parthenogenetic stem cells are similar to embryonic stem cells in their proliferation and multilineage in vitro differentiation capacity. The aim of this study is to derive multipotent neural stem cells from human parthenogenetic stem cells that are stable to passaging and cryopreservation, and have the ability to further differentiate into functional neurons. Immunocytochemistry, quantitative real-time PCR, or FACS were used to confirm that the derived neural stem cells express neural markers such as NES, SOX2 and MS1. The derived neural stem cells keep uniform morphology for at least 30 passages and can be spontaneously differentiated into cells with neuron morphology that express TUBB3 and MAP2, and fire action potentials. These results suggest that parthenogenetic stem cells are a very promising and potentially unlimited source for the derivation of multipotent neural stem cells that can be used for therapeutic applications.

Keywords: homozygous n neural differentiation n neural stem cells n parthenogenetic stem cells n pluripotent

Dmitry A Isaev1, Ibon Garitaonandia1, Tatiana V Abramihina1, Tatjana Zogovic-Kapsalis1, Richard A West2, Andrey Y Semechkin1, Albrecht M Müller3 & Ruslan A Semechkin*1

1International Stem Cell Corporation, Carlsbad, CA, USA 2Western Michigan University, Kalamazoo, MI, USA 3University of Würzburg, Würzburg, Germany *Author for correspondence: Tel.: +1 760 940 6383 Fax: +1 760 940 6387 [email protected]

part of

Research Article Isaev, Garitaonandia, Abramihina et al. In vitro differentiation of human parthenogenetic stem cells into neural lineages Research Article

38 Regen. Med. (2012) 7(1) future science group

same time, parthenogenetic cells are able to per-sist and proliferate in many tissues including the brain [9]. Cibelli et al. described the establish-ing of nonhuman primate Macaca fascicularis parthenogenetic stem cells called Cyno-1, and as proof of pluripotent state, Cyno-1 in vitro differentiation was performed, and neural derivatives, amongst others, were obtained [7]. Later, Sánchez-Pernaute et al. obtained dopa-mine neurons from Cyno-1 in vitro by means of directed differentiation, and demonstrated their effectiveness in Parkinson’s disease models using rats and monkeys [10]. Neural differentiation of hpSCs in vitro was shown by Revazova et al. and Harness et al. [1,2,8]. Despite these studies, human parthenogenetic NSCs maintained in culture still have not been obtained.

We report here for the first time that NSCs derived from hpSCs (hpNSCs) are able to main-tain proliferative and differentiation potential during cultivation and expansion, which enables practical amounts of neurogenic material to be obtained.

Materials & methods�n Human parthenogenetic stem cells

The hpSC lines LLC2P, LLC6P and LLC12PH [1,2], were maintained on mitomycin-C inacti-vated mouse embryonic fibroblasts (Millipore) feeder layer in embryonic stem medium: KDMEM/F12 (Life Technologies), supple-mented with 15% KSR (Life Technologies), 2 mM l-glutamine (GlutaMA X™-I, Invitrogen), 0.1 mM MEM nonessential amino acids (Life Technologies), 0.1 mM b-mercapto-ethanol (Life Technologies), penicillin/strepto-mycin/amphotericin B (100 U/100 µg/250 ng) (MP Biomedicals) and 5 ng/ml bFGF (Peprotech). Cells were passaged with dispase or collagenase IV (both Life Technologies) every 5–7 days with split ratio of 1:4 or 1:6.

�n Neural induction & NSCsNeural induction was done using an adher-ent culture system as described by Shin et al. [11] with modifications. Briefly, hpSCs main-tained on mouse embryonic fibroblasts feeder layers for 5 days were passaged with dispase (Life Technologies) on CELLstart™ (Life Technologies) coated 60-mm petri dishes. During the next 4 days colonies of hpSCs were cultivated in embryonic stem medium, fol-lowed by culture in neural induction medium. Neural induction medium was DMEM/F12 containing N2 supplement (Life Technologies), 0.1 mM MEM nonessential amino acids, 2 mM

l-glutamine (GlutaMAX-I, Life Technologies), antibiotic solution and 20 ng/ml of bFGF. The day of medium replacement was considered as day 0 of neural induction. The areas with well-formed rosettes of neuroepithelial cells were iso-lated mechanically, dissociated to single-cell sus-pensions using TrypLE™ (Life Technologies) and transferred into CELLstart-coated 24-well plates. Cells were expanded in StemPro® NSC SFM medium (Invitrogen), supplemented with 20 ng/ml bFGF and 20 ng/ml EGF (both from Peprotech). Further expansion of NSCs was performed on CELLstart-coated 60-mm petri dishes in StemPro NSC SFM medium supple-mented as described above. Cells were dissociated with Accutase® (Life Technologies) during pas-saging. H9 hESC-derived NSCs (in this paper referred to as hNSCs; GIBCO® Invitrogen) were maintained under the same conditions.

Spontaneous differentiation of NSCs was performed in Neurobasal® medium (Life Technologies), supplemented with B27 with-out retinol (Life Technologies), 0.1 mM MEM nonessential amino acids, 2 mM l-glutamine (GlutaMAX-I, Life Technologies) and antibiotic solution. For cryopreservation, NSCs were fro-zen in 10% DMSO in supplemented StemPro NSC SFM medium.

�n Real-time quantitative PCRTotal RNA was isolated using the QIAsymphony® automatic purification system, according to the manufacturer’s instructions (Qiagen). Then, 100–500 ng total RNA was used for reverse transcription with the iScript™ cDNA synthe-sis kit (Biorad). To analyze RNA quantities of specific genes, PCR reactions were performed in duplicate using 1/25th of the cDNA per reaction and the QuantiTect® Primer Assay together with QuantiTect SYBR Green master mix (Qiagen) (primers listed in Table 1). Reverse transcriptase real-time quantitative PCR (qRT-PCR) was performed using the Rotor-Gene® Q (Qiagen). Relative quantification was performed against a standard curve and quantified values were normalized against the input determined by PPIG. After normalization, the standard error of the mean was calculated (biological replica, n = 2–7). The results are presented as mean ± standard error of mean. Differences between data groups were determined using Student’s t-test at p ≤ 0.05.

�n ImmunocytochemistryFor immunostaining, NSCs were fixed with 4% paraformaldehyde for 10 min at room temperature


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and permeabilized by 0.1% Tween-20 and 0.3% Triton X-100 in phosphate buffered saline (PBS) for 1 h at room temperature. After permeabili-zation, the cells were blocked overnight at 4°C with 3% normal goat serum. Cells were then incubated overnight at 4°C with primary anti-bodies against Sox2 (1:100), Nestin (1:200) and Musashi-1 (1:300). Cells were washed three-times with PBS and incubated with secondary antibodies (1:500) for 2 h at room temperature. For one-step staining of differentiated neurons, antitubulin bIII Alexa Fluor 488 coupled anti-body was used according to the manufacturer’s instruction (Covance). The nuclei were stained with DAPI. The list of primary and secondary antibodies is given in Table 2.

�n Flow cytometryFor FACS analysis, NSCs were harvested with Accutase, washed with PBS and fixed with 4% paraformaldehyde overnight at 4°C. NSCs were washed with PBS and incubated with primary antibodies against Musashi-1 (1:40) in a solu-tion containing 0.1% Tween-20 and 1% normal goat serum in PBS for 1 h at room temperature. Cells were washed with PBS and incubated with secondary antibody conjugated to Alexa 488 (Table 2) for 1 h at room temperature. The samples were run on a Becton Dickinson FACSCalibur™ 4-color f low cytometer and the data were analyzed with CellQuest Pro™ software (v6.0).

�n ElectrophysiologyThe coverslips where the neurons were growing were cut into smaller segments to fit into the fusi-form test chamber sized 5 mm at the widest point and 1 cm long. The test chamber was perfused with Tyrodes solution containing 1.8 mM CaCl

2;

1 mM MgCl2; 4 mM KCl; 140 mM NaCl; 10 mM

glucose; 10 mM HEPES; 305–315 mOsm; pH 7.4 (adjusted with 5 M NaOH). Electrodes were prepared with 3–5 MOhms resistance when filled with 140 mM KCl; 10 mM MgCl

2; 6 mM

EGTA; 5 mM HEPES-Na; 5 mM ATP-Mg; 295–305 mOsm; pH 7.25 (adjusted with 1 M KOH). Data were processed with a 5 KHz Bessel filter and acquired at 10–20 KHz using a Multiclamp 700 A amplifier (Axon Intruments) and Pclamp software. All experiments were per-formed at room temperature under a microscope in a continuous flow chamber.

results�n Neural induction & neuroepithelium

generationThe overall objective of this study was to dem-onstrate that NSCs can be efficiently derived from hpSCs. For this purpose we have chosen the adherent model [11], since it provides a more uniform and synchronous formation of neuroec-toderm compared with the protocol using embry-oid bodies [12]. Unlike the original protocol [11], we did not use any feeder cells to grow stem cell

Table 1. real-time PCr primers.

Gene Catalog # Producer

ACTA1 QT00199815 QuantiTect® Primer Assay Qiagen

AFP (a-fetoprotein) QT00085183 QuantiTect® Primer Assay Qiagen

FOXD3 QT01018794 QuantiTect® Primer Assay Qiagen

FOXO4 QT00029141 QuantiTect® Primer Assay Qiagen

GFAP QT00081151 QuantiTect® Primer Assay Qiagen

MAP2 QT00057358 QuantiTect® Primer Assay Qiagen

MS1 (Musashi-1) QT00025389 QuantiTect® Primer Assay Qiagen

NES (Nestin) QT00235781 QuantiTect® Primer Assay Qiagen

OLIG2 QT01156526 QuantiTect® Primer Assay Qiagen

PAX6 QT00071169 QuantiTect® Primer Assay Qiagen

POU5F1 (OCT4) QT00210840 QuantiTect® Primer Assay Qiagen

SNAI2 (Slug) QT00044128 QuantiTect® Primer Assay Qiagen

SOX1 QT01008714 QuantiTect® Primer Assay Qiagen



TUBB3 (Tubulin-bIII) QT00083713 QuantiTect® Primer Assay Qiagen

PPIG (cyclophilin G) QT01676927 QuantiTect® Primer Assay Qiagen



colonies for neural induction. After 5 days on CELLstart, colonies of hpSCs appeared to be fully formed and did not have any morphologi-cal differences with colonies grown on feeders (Figure 1a).

After the medium was replaced, the first signs of neuroepithelial (NEP) rosettes appeared on the second day of neural induction. On the seventh day of neural induction, cell colonies appeared as large areas containing clusters of NEP rosettes. In these clusters most rosettes had well-formed lumen (Figure 1b). qRT-PCR analysis revealed that transcriptional activity of the key neuroectodermal genes PAX6 and SOX1 were increased at this stage in comparison with undifferentiated hpSCs, whereas pluripo-tency marker OCT4 was dramatically down-regulated (Figure 1C). The expression of specific neural marker genes NES and MS1 was also high. Endodermal marker AFP and mesodermal marker ACTA1 were not detected by qRT-PCR in the NEP rosettes (data not shown).

It is noteworthy that increased expres-sion of SOX1 and SOX3 was observed in the hpSC-derived NEP rosettes, whereas a slight decrease of SOX2 expression was found (Figure 1C), which might be associated with OCT4 down regulation [13].

In this study, development of NEP rosettes in hpSC colonies grown on CELLstart occurred within a week after replacement of embryonic stem medium with neural induction medium. The early formation of the neuroepithelium in our model may be a result of the initial absence of feeder and/or the influence of CELLstart on the growing hpSC colonies.

�n hpNSC culture & phenotypeThe isolation and dissociation to single-cell sus-pension of NEP rosettes resulted in the hpNSC proliferating cell population. hpNSCs were

passaged every 4–5 days at split ratio 1:2 over 5 months. hpNSCs maintained specific morphol-ogy similar to hNSCs for at least 30 passages (Figure 2a).

The expression of specific genes SOX2, NES and MS1 in hpNSCs was confirmed at the protein level by immunocytochemical detec-tion of corresponding antigens Sox2, Nestin and Musashi-1 (Figure 3). We also compared transcriptional activity of the marker genes in five hpNSC lines, three heterozygous and two homozygous, with the hNSCs derived from H9 hESCs. The relative expression levels of NES, MS1 and PAX6 in hpNSC lines were close to those in hNSCs. The expression of pluripo-tency marker OCT4 was at detectable but very low levels in all NSC lines and the endodermal marker AFP was not detected (Figure 2b).

We analyzed the expression of Musashi-1 by flow cytometry and found that more than 80% of the hpNSC population expresses Musashi-1 (Figure 2C), which is consistent with previ-ously published results on NSCs derived from hESCs [11].

To support NSC proliferation growth fac-tor bFGF is required, but this inhibits endog-enous SHH, leading to a rapid loss of ability to differentiate into neurons, and promotes metamorphosis of NSCs into neural crest ecto-mesenchymal cells [14]. The expression levels of neural crest marker gene FOXD3 and mesoder-mal marker ACTA1 were low in hpNSCs up to 30 passages, and even lower in comparison with hNSCs (Figure 2b). These data indicate the absence of large-scale metamorphosis of hNSCs into ectomesenchymal cells.

To demonstrate that hpNSCs can be pre-served, we froze and thawed hpNSCs. The hpNSCs had the same morphology and prolif-eration rate after cryopreservation. The hpNSCs also expressed similar levels of the neural markers

Table 2. Antibodies for immunostaining and FACs.

Antigen Catalog # Producer

Tubulin bIII A488-435L Covance

Sox2 ab92494 AbCam

Musashi-1 ab52865 AbCam

Nestin MAB5326 Millipore

Goat anti-mouse, -488 35503 ThermoScientific

Goat anti-rabbit, -488 35553 ThermoScientific

Goat anti-mouse, -549 35508 ThermoScientific

Goat anti-rabbit, -549 35558 ThermoScientific

Donkey anti-rabbit, -488 A-21206 Life Technologies



SOX2, NES, MS1 and PAX6 as determined by qRT-PCR (Figure 2D).

�n Differentiation of hpNSCsIn the B27-supplemented medium without growth factors bFGF and EGF, spontaneous differentiation of hpNSCs and hNSCs occurred within 4 weeks, resulting in the generation of predominantly neuron-like cells. Neuronal dif-ferentiation was confirmed by positive immu-nocytochemical staining for tubulin bIII and by high transcriptional activity of tubulin bIII encoding gene TUBB3 and associated MAP2, revealed by qRT-PCR analysis (Figure 4a & b). Transcriptional activity of specific oligodendro-cyte marker FOXO4 and astrocyte marker GFAP indicated the presence of glial derivatives among differentiated cells.

�n Electrophysiology of hpSC-derived neuronsIn the hpSC-derived neurons, action potentials were elicited by short (100–200 µs, 2–4 nA) bipolar current pulses, with an apparent thresh-old voltage for activation near -35 mV (Figure 4C), an overshoot to +40 mV and a 95% duration of 4 ms. In spite of the high threshold voltage, which could implicate calcium current-dominant action potentials, the dominant inward currents that were observed from these same cells under voltage-clamp mode were from fast sodium chan-nels. Figure 4D uses a voltage clamp waveform that hyperpolarizes the membrane to -100 mV to reset sodium channels, then steps to various sodium channel inactivating potentials between -90 and -45 mV to show the steady-state inac-tivation of sodium to a marginal test potential

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Figure 1. Generation of neuroepithelial rosettes from human parthenogenetic stem cells. (A) Colonies of human parthenogenetic stem cells (hpSCs) on CELLstart™, 4 days of cultivation, phase contrast, scale bar: 200 µm. (B) Neuroepithelial rosettes formed in the colonies of hpSC-1 on the seventh day of neural induction, phase contrast, scale bar: 100 µm. (C) Relative gene expression in hpSCs (dark blue bars) and in the neuroepithelial rosettes (light blue bars) on the seventh day of neural induction. Data represent mean ± standard error of mean.



to -45 mV instead of -5 mV. The most negative sodium test potential was intended to demonstrate the presence of sodium currents separately from the presence of calcium currents and at the same time provide minimal activation of the outward potassium currents.

Finally, the voltage was further stepped to vari-ous potentials between -120 and +60 mV to dem-onstrate the presence of any potassium currents, including inwardly rectifying (with activation at negative potentials), outwardly rectifying or transient outward currents (with activation above -40 mV). In addition, this last family of voltages will also activate any inward calcium currents since

they will not become inactivated by voltages below -45 mV. This waveform, as illustrated in Figure 4D below the family of current recordings, revealed very little inward calcium current, but strong inward currents greater than 2 nA consistent in voltage dependence and temporal profile with fast sodium channels, and a quickly activating then slowly inactivating similarly sized outward cur-rent consistent with the voltage dependence and temporal profile of A-type potassium channels.

discussionThe main purpose of this study was to derive NSCs from hpSC lines. We successfully demonstrated

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Figure 2. Phenotype of neural stem cells derived from human parthenogenetic stem cells. (A) Neural stem cells derived from hpNSCs in adherent culture, phase contrast, scale bar: 50 µm. (B) Relative transcriptional activity levels of important genes in heterozygous hpNSCs (dark blue bars), homozygous hpNSCs (light blue bars) and H9 human embryonic stem cell-derived neural stem cells (red bars). (C) Differentiation efficiency of human parthenogenetic stem cells into hpNSCs determined by FACS. hpNSC culture is more than 80% Musashi-1 positive. (d) Relative transcriptional activity levels of hpNSC before (dark blue bars) and after cryopreservation (light blue bars). Data represent mean ± standard error of mean. hpNSC: Neural stem cells derived from human parthenogenetic stem cells.



that pluripotent hpSCs can serve as a reliable source of NSCs. The hpNSCs obtained are capa-ble of relatively stable proliferation while main-taining their neurogenic potential and ability to provide sufficient quantity of cells for cryopreser-vation and further study. We demonstrated that these hpNSCs express the same markers as NSCs derived from hESCs. We observed differences in gene expression between the homozygous and heterozygous hpNSC lines that could be due to differences between the original stem cell lines, or due to small variations in the derivation pro-cedure. Further research would be required to understand these differences in gene expression.

The differences observed between transcrip-tion activity of SOX1, SOX2 and SOX3 could be explained by the fact that these genes are mem-bers of SOXB1 subgroup of transcription factors whose exact role in the maintenance of neural progenitors and in restriction of their ability to

differentiate still remains unclear. It was shown that the functions of these genes are redundant [13,15], thus it is possible that in maintaining the properties of NSCs, SOXB1 genes mutually compensate each other.

Based on gene expression data, the hpNSCs can be considered as precursors of all three main types of neural derivatives. At the same time, the expression of oligodendrocyte marker FOXO4 was higher in the differentiated neurons from hpNSCs compared with neurons derived from hNSCs. This finding correlated with higher expression of transcriptional factor OLIG2 spe-cific for oligodendrocyte and neuron precursors (Figure 2b).

The hpNSCs are able to further differenti-ate in vitro into neurons that express appro-priate neuronal markers and are shown to be functional as demonstrated via electrophysi-ology. Taken together these results suggest

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Figure 3. Neural stem cells derived from human parthenogenetic stem cells stained for Nestin, sox2 and Musashi-1. Scale bars: 20 µm.



that hpNSCs could be a reliable source in the future for the treatment of neurodegenerative diseases.

ConclusionThe advantage of hpSCs is their ability to immune match millions of patients. hpSCs also overcome the ethical hurdle of hESCs by avoid-ing the destruction of viable human embryos. The successful derivation of NSCs from homozygous hpSCs represents a unique source of immunogenic stem cells for the treatment of multiple neurodegenerative diseases.

Financial & competing interests disclosureDA Isaev, I Garitaonandia, TV Abramihina, T Zogovic-Kapsalis, AY Semechkin and RA Semechkin are employ-ees and stockholders of International Stem Cell Corporation (ISCO). RA West is ISCO’s independent contractor and an employee of West Labs Scientific. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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Figure 4. spontaneous differentiated neural stem cells derived from human parthenogenetic stem cells. (A) During 4 weeks neural stem cells derived from human parthenogenetic stem cells (hpNSCs) differentiated mainly into tubulin bIII-positive (green) neurons; scale bar 10 µm. Nuclei are blue, DAPI. (B) Neuronal markers TUBB3 and MAP2 and glial markers GFAP and FOXO4 expression in spontaneously differentiated hpNSCs (dark blue bars) and human neural stem cells (light blue bars). (C) Whole-cell electrophysiology of hpNSC-derived neurons. Action potentials were elicited from three cells, producing 4 ms action potentials with overshoot to +40 mV. (d) A multistage waveform, shown below the family of traces with numbers representing membrane potentials, reveals the major inward and outward currents present in the cells under voltage clamp conditions, showing (1) an inward fast sodium current, (2) a lack of a large inward calcium current, (3) a large transient outward current and (4) a lack of inwardly rectifying potassium current.



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executive summary

� Human parthenogenetic stem cells (hpSCs) are derived from the inner cell mass of blastocysts obtained from unfertilized oocytes.

� hpSCs can be either heterozygous or homozygous depending on the way the genome forms from the maternal chromosome set. The differentiated derivatives of homozygous hpSCs with common HLA types can match millions of individuals.

� In this study, neuroepithilial rosettes were derived from both homozygous and heterozygous hpSCs through an adherent model under feeder-free conditions.

� Human parthenogenetic neural stem cells (hpNSCs) were obtained through isolation and dissociation into single-cell suspensions of neuroepithilial rosettes. hpNSCs expressed PAX6, SOX1, SOX2, NES and MS1, and very low levels of OCT4. hpNSCs can be cryopreserved and maintained in culture for up to 30 passages.

� hpNSCs can be further differentiated into neurons and glia, and the hpNSC-derived neurons express neuronal markers and are functional.

ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the princi ples outlined in the Declaration of Helsinki for all

human or animal experimental investigations. In addi-tion, for investi gations involving human subjects, informed consent has been obtained from the participants involved.

in vitro differentiation of human parthenogenetic stem...

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