Published Ahead of Print on May 13, 2015 as 10.5966/sctm.2014-0289.
Tissue-Specific Progenitor and Stem Cells
TISSUE-SPECIFIC PROGENITOR AND STEM CELLS Direct Conversion of Cord Blood CD34+ Cells Into Neural Stem Cells by OCT4
Key Words. Direct conversion x Neural stem cell x Cord blood x OCT4 Departments of aPathology, b Surgery, and cObstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; d Department of Medicine, Loma Linda University, Loma Linda, California, USA Correspondence: Wenbin Liao, M.D., Ph.D., Department of Pathology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York 11794, USA. Telephone: 949-824-7298; E-Mail: vwliao@ gmail.com; or Yupo Ma, M.D., Ph.D., Department of Pathology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York 11794, USA. Telephone: 631-444-2214; E-Mail: yupo.ma@ stonybrookmedicine.edu Received December 11, 2014; accepted for publication April 8, 2015. ©AlphaMed Press 1066-5099/2015/$20.00/0 http://dx.doi.org/ 10.5966/sctm.2014-0289
ABSTRACT Cellular reprogramming or conversion is a promising strategy to generate desired stem cell types from somatic cells. Neural stem cells (NSCs) have the potential to regenerate central nervous system tissue and repair damage in response to injury. However, NSCs are difficult to isolate from human tissues and expand in sufficient quantities for therapy. Here, we report a method to generate neural stem cells from cord blood CD34-positive cells by ectopic expression of OCT4 in a feeder-free system. The induced cells (iNSCs) show a characteristic NSC-like morphology and can be expanded in vitro for more than 20 passages. In addition, the iNSCs are positive for neural stem cell-specific markers such as Nestin and Musashi-1 and are similar in gene expression patterns to a human neural stem cell line. The iNSCs express distinct transcriptional factors for forebrain, hindbrain, and spinal cord regions. Upon differentiation, the iNSCs are able to commit into multilineage mature neural cells. Following in vivo introduction into NOD/SCID mice, iNSCs can survive and differentiate in the mouse brain 3 months post-transplantation. Alternatively, we were also able to derive iNSCs with an episomal vector expressing OCT4. Our results suggest a novel, efficient approach to generate neural precursor cells that can be potentially used in drug discovery or regenerative medicine for neurological disease and injury. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:1–9
SIGNIFICANCE This study describes a novel method to generate expandable induced neural stem cells from human cord blood cells in a feeder-free system by a single factor, OCT4. The data are promising for future applications that require the generation of large amounts of autologous neural stem cells in disease modeling and regenerative medicine.
INTRODUCTION Neural stem cells (NSCs) can generate the vast array of neurons, astrocytes, and oligodendrocytes that comprise the nervous system. Consequently, NSCs hold significant potential as a therapeutic strategy for treatment of spinal cord injury, as well as neurodegenerative diseases such as Parkinson’s disease [1]. In the past, the use of NSCs for the treatment of neurological disease was hindered by limited fetal and adult NSC sources. Recent advances in cellular reprogramming allowed for the generation of induced pluripotent stem cells (iPS) and somatic stem cells from various somatic tissues [2–6]. To that effect, NSCs can be obtained by differentiation from iPS cells [7, 8]. However, differentiating iPS cells into NSCs presents with numerous drawbacks that need to be addressed before clinical applications: prolonged culture time, putative low reprogramming efficiency, and tumorigenic risk because of the possible presence of residual pluripotent cells
postdifferentiation [9]. An alternative and safer strategy may be to bypass the pluripotent state and convert directly to NSCs. Indeed, several groups have described successful direct conversion from fibroblasts or other somatic cells to NSCs [4, 10–12]. However, the generation of NSCs required the use of feeder cells, a potential limitation for any cell therapy-based application. In our study, we describe the generation of expandable NSCs from human cord blood CD34-positive cells using a novel method that is efficient, does not require feeder cells, and is mediated by a single factor, OCT4. We also describe a nonintegrating, episomal vector for OCT4-mediated generation of induced NSCs from cord blood.
MATERIALS AND METHODS Cell Culture Cord blood (CB) CD34+ cells were isolated using human CD34+ labeling kits (Miltenyi Biotec,
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WENBIN LIAO,a NICK HUANG,a JINGXIA YU,a ALEXANDER JARES,a JIANCHANG YANG,b GARY ZIEVE,a CECILIA AVILA,c XUN JIANG,a XIAO-BING ZHANG,d YUPO MAa
Published Ahead of Print on May 13, 2015 as 10.5966/sctm.2014-0289.
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Direct Conversion of CD34+ Cells Into NSCs by OCT4 Lentivirus and Episomal Vector Lentiviruses carrying human OCT4 gene or control green fluorescent protein (GFP) gene under spleen focus-forming virus (SFFV) promoter were packaged and produced using the 293FT cell line. The viruses were concentrated by centrifugation and stored at 280°C. Fresh isolated or frozen human CD34+ cells were cultured in growth medium for 24 hours before transduction. Lentiviruses were added at a multiplicity of infection of 2–5 in the presence of 8 mg/ml Polybrene (Millipore) for 4–6 hours (day 0). Then transduced cells were washed in warm phosphatebuffered saline (PBS) and seeded at a density of ∼20,000 cells per mm2 in tissue culture plates that were precoated with 10 mg/ml fibronectin. The next day (day 1), half of the growth medium was removed, and the same amount of Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (DMEM/DF12 supplemented with 15% FBS, 10 ng/ml human bFGF, and 100 U/ml penicillin/streptomycin) was added. On day 2, the medium was changed to DMEM/F12 medium completely and refreshed every 2 days. On day 7, the adherent cells reached confluence and were replated at a density of 50,000 cells per mm2 after Accutase (Millipore) digestion. When the cells were confluent again, all cells were dissociated into single cells and plated at a density of 50,000 cells per mm2 on laminin-coated (20 mg/ml) plates in ReNcell medium. Routinely, the induced cells (CB-iNSCs) were passaged every 3–4 days with a doubling time of ∼24 hours. For neurosphere formation, the CBiNSCs were seeded in low attachment Petri dishes at a density of 200,000 cells per ml in ReNcell medium. During the cellular conversion procedure, 3 mM CHIR99021 (Stemgent, Cambridge, MA, https://www.stemgent.com) was used starting from the time of lentiviral transduction until the second passage in ReNcell medium. ReNcell medium was changed every 2–3 days. For conversion of CD34+ cells to iNSCs by OCT4 episomal vector (eiNSCs), the CD34+ cells were transfected with EBNV1based pCEP4-OCT4 expression plasmid by electroporation using a human CD34 cell nucleofector kit (Lonza, Koln, Germany, http://www.lonza.com). Cell culture and media changes after transfection were similar to those for the lentivirus-based cellular conversion, except for the fact that hypoxic conditions (5% O2) were required to generate eiNSCs. 3-Deazaneplanocin (Dzep; Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich. com) was added to the solution after the first passage in ReNcell medium.
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Differentiation of CB-iNSCs For induction of CB-iNSCs to three neural cell lineages, CB-iNSCs were cultured in poly-L-ornithine/laminin-coated glass coverslips in ReNcell medium without bFGF and EGF for 14 days, which allowed random differentiation and maturation of CB-iNSCs. For specific differentiation into neural cells, cells were induced by addition of 5 mM forskolin (Sigma), 20 ng/ml brain-derived neurotrophic factor, and 20 ng/ml glial cell-derived neurotrophic factor (GDNF; PeproTech, Rocky Hill, NJ, https://www.peprotech.com). For oligodendrocyte differentiation, CB-iNSCs were stimulated with 100 ng/ml Sonic hedgehog (SHH) for 4 days and then cultured in DMEM/F12 supplemented with 2% B27, 10 ng/ml platelet-derived growth factor AA (Peprotech), and 40 ng/ml thyroid hormone T3 (Sigma) for 3 weeks with half medium change twice each week. For dopaminergic neuron differentiation, CB-iNSCs were induced by human 100 ng/ml FGF-8 and 100 ng/ml SHH for 3 days and by 20 ng/ml ciliary neurotrophic factor and 25 ng/ml GDNF for 2 weeks.
Electrophysiology Whole-cell patch clamp was performed to measure the membrane current in single cells from undifferentiated or neurally differentiated CB-iNSCs or hcx NSCs. The cells were replated on polylysine-coated coverslips 1 day before the assay. The patch pipette resistances were 1–3 MV before sealing. The pipette solution contained (in mmol/l) 50 KCl, 80 potassium aspartate, 1 MgCl2, 3 Mg-ATP, 10 EGTA, and 10 HEPES (pH adjusted to 7.2 with KOH). The extracellular solution contained (in mmol/l) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH adjusted to 7.4 with NaOH). Voltage and current signals were recorded by the amplifiers (model Axopatch-1B; Axon Instruments/Molecular Devices Corp., Union City, CA, http://www.moleculardevices.com), digitized (model DIGIDATA 1320A digitizers; Axon Instruments/ Molecular Devices Corp.), and finally transferred to a personal computer.
Regular PCR and Quantitative PCR Total RNA was extracted with an AllPrep DNA/RNA mini kit (Qiagen, Hilden, Germany, http://www.qiagen.com), and cDNA was synthesized using a QuantiTect reverse transcription kit (Qiagen). Polymerase chain reaction (PCR) amplification was conducted with Platinum PCR Supermix, high fidelity (Life Technologies, Grand Island, NY, http://www.lifetechnologies.com). Quantitative PCR (qPCR) was run on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) with Power SYBR Green PCR Master Mix (Life Technologies). All qPCRs were conducted in triplicate. The expression results of real-time PCR were presented by log2fold according to D(DCt), which is normalized to b-actin expression. The detailed information for all primers is provided in supplemental online Table 2.
Gene Expression Microarray Total RNAs of CB CD34+ cells, CB-iNSCs, and hcx NSCs were extracted using the kits as mentioned above. RNA quantity and quality (2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA, http://www.agilent.com) was determined to be optimal before further processing. The Affymetrix (Santa Clara, CA, http://www. affymetrix.com) human HG-U133plus2 GeneChip arrays hybridization, staining, and scanning were performed using Affymetrix standard protocols as previously described by the Stony Brook S TEM C ELLS T RANSLATIONAL M EDICINE
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Auburn, CA, http://www.miltenyibiotec.com) and cultured in Stemspan medium (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) supplemented with 10% fetal bovine serum (FBS), 100 ng/ml human stem cell factor, 100 ng/ml human thrombopoietin, 100 ng/ml human fms-like tyrosine kinase 3 ligand (Peprotech, Rocky Hill, NJ, http://www.peprotech.com), and 100 U/ml penicillin/streptomycin (Gibco, Grand Island, NY, http://www. invitrogen.com). A human cortex (hcx) neural progenitor cell line was purchased from Millipore (Billerica, MA, http://www. millipore.com) and maintained in ReNcell medium: ReNcell NSC maintenance medium (Millipore) supplemented with 20 ng/ml human epidermal growth factor (EGF), 20 ng/ml human basic fibroblast growth factor (bFGF) (Peprotech), and 100 U/ml penicillin/ streptomycin.
Inducing Neural Stem Cells From Cord Blood by OCT4
Published Ahead of Print on May 13, 2015 as 10.5966/sctm.2014-0289.
Liao, Huang, Yu et al.
University DNA Microarray Core Facility. All genes of neurogenesis and hematopoiesis according to Gene Ontology terms (AmiGO, http://www.geneontology.org) were analyzed, and upregulation or downregulation fold changes were normalized to CB CD34+ cells. The heat map of gene expression levels was generated by R software.
Transplantation of CB-iNSCs Into Mice
Immunostaining Cultured cells or brain sections were fixed in 4% paraformaldehyde for 15 minutes at room temperature and washed with PBS. Nonspecific antibody binding was blocked by using 1% bovine serum albumin for 30 minutes, and cells were permeabilized with 0.3% Triton X-100 (Sigma) in PBS (PBS-T) for 30 minutes at room temperature. Cells were rinsed and then incubated in primary antibody containing 0.1% overnight at 4°C. After washing in PBS, the cells were incubated in secondary antibody for 1 hour at room temperature. The cells were immunostained with the following anti-human primary antibodies: anti-Nestin, anti-Musashi1, anti-tyrosine hydroxylase (TH), anti-bIII tubulin (Tuj1), anti-glial fibrillary acidic protein (GFAP), and anti-29,39-cyclic nucleotide 39-phosphodiesterase (CNPase). Primary antibodies were detected with the phosphatidylethanolamine- or fluorescein isothiocyanate-conjugated secondary antibody. 49,6-Diamidino-2phenylindole (DAPI) was used to identify cell nuclei. Stained cells were preserved in antifading mount solution that contained DAPI. Stained cells were examined and photographed under an EOVS fluorescent microscope (Life Technolgies).
Statistical Analysis The results are presented as mean 6 SEM. Two-tailed Student’s t test was performed for comparison. p , .01 was considered statistically significant.
RESULTS Conversion of Cord Blood CD34+ Cells Into Neural Stem Cells in a Feeder-Free System Previously, human cord blood cells were reported to contain cells with neurogenic properties [13–15]. In order to detect whether
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there are any neural precursor cells in the CD34+ fraction of CB, we cultured CB CD34+ cells directly on laminin-coated tissue culture plates in ReNcell medium in the presence of human EGF and bFGF, which is a well established and standard culture condition for human neural stem cells (Millipore). In repeated attempts, we did not obtain any adherent cells resembling NSCs. In addition, we failed to detect the expression of any of the well-known neural stem cell genes in CB CD34+ cells by PCR (supplemental online Fig. 1B). Flow cytometry analysis further confirmed the absence of Nestin in the CB CD34+ cells (supplemental online Fig. 1C). These results suggest the absence of neural precursor cells in the original CD34+ cell population. We transduced cord blood CD34+ cells with lentiviruses carrying OCT4 or control GFP gene driven by an SFFV promoter. After OCT4 transduction in human cord blood CD34+ cells, adherent spindle-like cells could be observed as early as 24 hours posttransduction. When cultured in medium containing human bFGF, the derived adherent cells exhibited high proliferation potency (Fig. 1). In contrast, in the control GFP vector transduction, although a few adherent cells were detected in culture posttransduction, none displayed proliferative properties. Additionally, after multiple attempts with transduction with OCT4, cord blood CD342 cells did not generate proliferating adherent cells. In our system, the adherent cells in OCT4 transduced CD34+ CB groups consistently reached confluence 6–8 days after transduction, at which point, the number of adherent cells was ∼10-fold the number of the initial CD34+ cells. Consequently, at passage 1, we obtained ∼100-fold the number of induced NSCs (CB-iNSCs) relative to the initial CD34+ cell number (supplemental online Table 1). These CB-iNSCs were highly expandable (.20 passages, .2 months in culture) with unchanged morphology and stable doubling times (24–36 hours) in vitro (Fig. 1). In order to determine whether iPS cells were initially derived and subsequently programmed to neural stem cells in the culture, we stained the adherent cells at different time points (days 3 and 6 after OCT4 transduction) with TRA-1-60, TRA-1-81, and SSEA-3/4; none of these surface markers positively reacted with the cells (data not shown). In addition, we did not see teratoma formation after subcutaneous injection of CB-iNSCs in NOD/SCID mice (supplemental online Table 3). Thus, the data suggested that iNSCs were directly converted from CD34+ cells without a pluripotent stem cell intermediate step.
Characterization of CB-iNSCs The CB-iNSCs presented with characteristic NSC-like morphology. Subsequent immunostaining revealed that CB-iNSCs expressed neural stem cell markers such as Nestin, Musashi-1 (MSI1), and Pax6 (Fig. 2A). We also quantified the gene expression levels of CB-iNSCs by real-time PCR (Fig. 2B). As compared with the original cord blood cells, the neural stem cell markers Nestin, MSI1, Sox1, and Pax6 were significantly upregulated, whereas the hematological markers CD34, CD38, and CD45 were markedly downregulated. The gene expression patterns of neural stem cells and hematopoietic markers are similar between CB-iNSCs and hcx NSCs as compared with CB cells. Of note is that the overall gene expression levels were elevated in hcx NSCs for both neural stem cells and hematopoietic markers. This may be due to the ectopic expression of oncogene c-MYC in the hcx NSCs line. Further analysis using gene expression arrays confirm that CB-iNSCs are similar to hcx NSCs in gene expression patterns in neurogenesis and hematopoiesis (1578 genes in total) (Fig. 2C; supplemental online Fig. 2; supplemental online Tables 4, 5). We also compared the
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To track the CB-iNSCs in vivo, CB-iNSCs were labeled by transduction of GFP lentivirus before injection to the right striatum of NOD/SCID mice. Under anesthesia, the right side of the mouse skulls were exposed after tissue separation. A hole with a diameter of approximately 1.0 mm was drilled at center of the coordinates: anterior-posterior (AP): 0 mm; medial-lateral (ML): 2.5 mm. Then NOD/SCID mice were placed under stereotaxic apparatus and received 200,000 CB-iNSCs (in 2 ml of PBS) at the coordinates: AP: 0 mm; ML: 2.5 mm; dorsal-ventral: 3.5 mm. The cell injection was finished in 5 minutes, and the syringe was removed after another 5 minutes. Antibiotics (0.5% Bactrim in water) were administered postsurgically to the animals for 2 weeks. The cryostat sections (10 mm) of the animals’ brains were prepared after intracardiac perfusion, fixation, and dehydration 1 or 3 months after transplantation. The density of migrated cells in the contralateral hemisphere (left brain) was analyzed by counting GFP+ cells in six random fields of the coronal sections (AP: 0 mm) under a 310 lens of the fluorescent microscope.
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Inducing Neural Stem Cells From Cord Blood by OCT4
gene expression pattern of early and late passages CB-iNSCs and found no obvious differences between the results of passage 3 and passage 10 CB-iNSCs (Fig. 2B), suggesting maintained stem cell signature even during prolonged in vitro culture. Additionally, CB-iNSCs could form neurospheres in low-adherent culture dishes (Fig. 2D). Therefore, CB-iNSC gene expression profiles and morphology are highly consistent with neural stem cells.
Differentiation of CB-iNSCs In Vitro We induced the differentiation of the CB-iNSCs by simple removal of growth factors using a series of cytokines and chemicals. When the growth factors (EGF and bFGF) were removed from the ReNcell medium, the growth speed of CB-iNSCs largely slowed down, and the cells underwent differentiation into more long neurites, which increased in number and further matured with additional culture time (Fig. 3A; supplemental online Fig. 3). Synapse-like structures between cells could be seen after 2 weeks of in vitro differentiation. This is consistent with positive synapsin1 expression detected by PCR assay (Fig. 3C, 3D). By immunostaining, we found expression of Tuj1, GFAP, or CNPase in the differentiated cells, indicating the multilineage differentiation capacity of CBiNSCs (Fig. 3B). This was also confirmed by PCR results, in which markers of all three lineages of neural cells were detected: Tuj1, MAP2, and neurofilament (NF) for neurons; S100B for astrocytes and MBP; and CNPase for oligodendrocytes (Fig. 3D). In addition, we saw expression of both vesicular glutamate transporter 1 (VGLUT1) and g-aminobutyric acid (GABA) receptor in the differentiated cells, suggesting that CB-iNSCs could generate both excitatory (glutamatergic) and inhibitory (GABAergic) neurons. In an electrophysiological assay, we found that patterns of sustained potassium currents in response to stepwise voltage stimulations were similar between CB-iNSCs and hcx NSC-derived neural cells,
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suggesting similar functional properties across these two cell types (Fig. 3G).
Regional Pattern of CB-iNSCs In the development of the neural system, there are distinct expressions of transcriptional factors in the precursors for forebrain, midbrain and hindbrain regions. We detected forebrain (FOXG1), hindbrain (HOXA2, GBX1, EGR2, and HOXB2), and also spinal cord (HOXB6) markers (Fig. 3E), but not midbrain markers (PAX2 and EN1) in our CB-iNSCs (data not shown). Interestingly, we observed the same gene expression profile in passage 3 and passage 10 CB-iNSCs, suggesting that forebrain and hindbrain region-specific markers did not change over time (Fig. 3E). Control hcx NSC expressed forebrain (FOXG1) and hindbrain (GBX1, EGR2 and HOXB2) markers (Fig. 3E) and did not express midbrain markers (data not shown). The original CB CD34+ cells did not express any of the all midbrain and forebrain markers tested and were negative for hindbrain GBX1 as well. Notably, CB cells are positive for EGR2, HOXA2, HOXB2, and HOXB6 makers because they are also related to adult hematopoiesis [16, 17]. To check whether the CB-iNSCs could be induced for midbrain neuron commitment, we induced these CB-iNSCs in specific medium condition for midbrain dopaminergic neuron differentiation. The results showed that TH+/Tuj1+ positive cells could be detected after 2 weeks of induction (Fig. 3F). These data demonstrate that CB-iNSCs are readily expressing genes of forebrain, hindbrain, and spinal cord regions and can also be patterned to midbrain fate after specific stimuli.
CB-iNSCs Engraftment in Mouse Brain We labeled the CB-iNSCs with GFP marker and injected them into NOD/SCID mouse striatum under stereotaxic surgery. We found S TEM C ELLS T RANSLATIONAL M EDICINE
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Figure 1. Conversion of CB CD34+ cells to iNSCs by lentiviral OCT4 overexpression. (A): Diagram of the cellular conversion procedure. (B): Adherent appearance in OCT4 and control group. (C): In vitro long-term expansion of CB-iNSCs. (D): Growth curve of CB-iNSCs (n = 4). Scale bars = 50 mm. Abbreviations: bFGF, basic fibroblast growth factor; CB, cord blood; EGF, epidermal growth factor; hCB, human cord blood; iNSC, induced neural stem cell; Pn, passage n; SCF, stem cell factor; TPO, thrombopoietin.
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that a large proportion of these cells still survived in the brain 1 month after transplant (Fig. 4A). Long neurites could be detected from some of the cells. By immunostaining, we found Tuj1-, GFAP-, or CNPase-expressing cells from the GFP-positive injected cells, suggesting the in vivo maturation of CB-iNSCs (Fig. 4C). The injected cells are still observed 3 months post-transplantation (Fig. 4A). The cell number at the injection sites at 3 months is less than at 1 month post-transplantation (Fig. 4A); however, we found more cells in regions other than the injection site
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(Fig. 4B, 4D; supplemental online Fig. 4). We also detected Tuj1 expression of the injected cells 3 months post-transplantation (Fig. 4E). These data suggest that CB-iNSCs can survive, mature, and migrate long term in an in vivo setting. We also monitored the mice after receiving CB-iNSCs for long-term safety, and no abnormalities were observed more than 3 months postinjection (supplemental online Table 3). Therefore, CB-iNSCs are able to engraft and differentiate in the central nervous system without tumor formation.
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Figure 2. Characterization of CB-iNSCs. (A): Nestin, MSI1, PAX6 expression of CB-iNSC1, CB-iNSC3, and hcx NSC. (B): Relative expression of neural stem cells or hematopoietic stem cell genes of CB-iNSCs to CB CD34+ cells as quantitated by real-time polymerase chain reaction analysis. (C): Heat map of expression of genes in neurogenesis and hematopoiesis (a total of 1,578 genes) of CB-iNSCs and hcx NSCs by gene expression microarray. The expression levels of CB-iNSCs and hcx NSCs were presented in Log2fold change after being normalized to CB cells. (D): Neurosphere formation of CB-iNSCs. Scale bars = 50 mm. Abbreviations: CB, cord blood; DAPI, 49,6-diamidino-2-phenylindole; hcx, human cortex; iNSC, induced neural stem cell; MSI1, Musashi-1; NSC, neural stem cell; Pn, passage n.
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Inducing Neural Stem Cells From Cord Blood by OCT4
Generation of Integration-Free NSCs With an Episomal Vector Although direct conversion using lentivirus does provide an increasingly popular approach that bypasses the iPS cell state, there is the risk that random integration of foreign DNA into the host’s genome would potentially lead to insertional mutagenesis. To that effect, we sought to generate iNSCs from CB using an integration-free method. CB CD34+ cells were electroporated with an episomal vector containing a high expression OCT4 gene (Fig. 5A). After nucleofection, we successfully generated neural stem cells within 2–3 weeks (Fig. 5B), which we hereafter refer to as eiNSCs for brevity. iNSCs and eiNSCs are identical morphologically, and eiNSCs did not differ in the expression of typical NSC markers of Nestin and Musashi (Fig. 5C). In addition, eiNSCs are able to differentiate into neural and glial cells because neuron and astrocyte markers were observed after random differentiation (Fig. 5D). However, eiNSCs are slower in growth rate with a doubling time of approximately 36 hours, whereas iNSCs have a doubling time of approximately 24 hours. Also, eiNSCs failed to maintain long-term expansion in vitro because no growth was observed after three to four passages. When Dzep, a compound that was recently reported to increase the efficiency of iPS generation [18], was added to the culture, eiNSCs grew for two additional passages (Fig. 5B).
DISCUSSION OCT4 is a key transcriptional factor in the reprogramming of somatic cells to iPS cells [2, 3]. Our findings establish that the
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introduction of OCT4 alone is enough to directly convert mesoderm-originated cells (human cord blood derived CD34+ cells) to neuroectoderm cells without a pluripotent intermediate. Our method is a rapid and efficient approach to obtain highly expandable iNSCs that are able to differentiate into all three neural lineage cells. Given that the initiating cord blood cells grow as a suspension culture and that induced NSCs grow as adherent tissue culture cells, cellular conversion events are easy to detect and track. Furthermore, the iNSC adherent cells proliferate rapidly during cellular conversion. By passage 1, that is, within 2 weeks of conversion, we obtained an iNSC number that is a 100-fold increase relative to the starting CD34+ population. Crucially for therapeutic applications, the iNSCs are consistently able to maintain stable long-term expansion in vitro. Therefore, a single cord blood sample can yield a large amount of autologous iNSCs for use in drug screenings or regenerative cell therapy. The OCT4 expression level determines in part whether pluripotent stem cells maintain stemness or head to differentiation. In our study, sufficient Oct4 expression level is critical for successful CB-iNSCs generation. For instance, when Oct4 is driven by a CMV promoter, OCT4 expression is 50% lower than for the SFFV-Oct4 vector. As a result, lentiviral transduction of OCT4 driven by CMV promoter is not sufficient to convert CB cells into iNSCs. This is especially salient in a single-factor cellular reprogramming or conversion system. In previous reports, iNSCs were generated from a variety of cell sources [19–22]. However, translation into cell therapy applications, for which large amounts of cells are needed, was hindered by the limited proliferative potential of the derived iNSCs. Subsequent attempts by several groups generated more S TEM C ELLS T RANSLATIONAL M EDICINE
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Figure 3. In vitro differentiation of CB-iNSCs. (A): Differentiated CB-iNSCs show neuron-like morphology with long neurites as compared with undifferentiated cells. (B): Multilineage differentiation of CB-iNSCs as shown by staining of neuron (Tuj1), astrocyte (GFAP), or oligodendrocyte (CNPase) marker immunostaining. (C): Synapse-like structure in differentiated CB-iNSCs. (D): Polymerase chain reaction analysis of genes related to neurotransmitter and multilineage neural cells. i: Undifferentiated CB-iNSC 1. ii: Differentiated CB-iNSC 1. iii: Differentiated CB-iNSC 3. (E): Regional pattern of CB-iNSCs. (F): Dopaminergic neural cell differentiation from CB-iNSCs. (G): Representative voltage-clamp recordings of potassium channel currents in response to stepwise voltage stimulations (2120 mV to +120 mV) for neurally differentiated cells from CB-iNSCs and control hcx NSCs. Scale bars = 50 mm. Abbreviations: CB, cord blood; DAPI, 49,6-diamidino-2-phenylindole; GABA, g-aminobutyric acid; GFAP, glial fibrillary acidic protein; hcx, human cortex; iNSC, induced neural stem cell; NF, neurofilament.
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expandable iNSCs using combinatorial expression of a set of transcriptional factors [4, 10, 12]; however, obstacles to cell therapy remained. Those iNSCs retained restricted differentiation potential and the use of oncogenic factors, such as c-MYC, highlighted the risks of long-term constitutive expression of reprogramming transgenes after the target tissue had already been derived. Indeed, a recent study reported the generation of iNSCs from cord blood CD45+/CD133+ cells with SOX2 alone and with the addition of c-MYC. Another separate study reported the generation of iNSCs from fibroblasts using SOX2 alone [11, 23]. In the first study [23], the iNSCs from cord blood cells showed restricted neural lineages immediately after conversion, and prolonged cell culture in permissive conditions is required to expand and further dedifferentiate the induced neuronal cells to an earlier cell stage. In the second study [11], SOX2 was used to convert mouse or human fibroblasts into neural precursor cells; however, there remain no in vivo data on human iNSCs. Additionally, in both of these studies, feeder cells were required for conversion. In contrast, we were able to consistently generate CB-iNSCs without support from a feeder
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cell system. We also attempted to convert human cord blood CD342 cells or human skin fibroblasts into NSCs with the feeder-free system; however, no iNSCs were successfully obtained (data not shown). Thus, CD34+ cells are an easily obtainable and efficient source for patient-specific NSC conversion. CB-iNSCs are able to give rise to multilineage neural cells both in vitro and in vivo. Similar to NSCs isolated from neural tissues, CB-iNSC expansion is dependent on stimulation with human EGF and bFGF and the removal of growth factors that lead to cell maturation. Intriguingly, CB-iNSCs show multiple regional patterns including forebrain, hindbrain, and spinal cord, and these patterns are maintained at least for 10 passages. We also found that FGF-8/SHH stimulation could induce the generation of midbrain-specific cells. Importantly for long-term translational applications, CB-iNSC consistently maintained differentiation capacity in vivo. In addition, the CB-iNSCs are able to survive at least 3 months without any aberrant proliferation in the mouse brain, suggesting long-term engraftment and safety in vivo.
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Figure 4. In vivo transplantation of cord blood (CB)-induced neural stem cells (iNSCs). (A): The GFP-labeled CB-iNSCs (green) are visible in the injection region (right striatum, marked ellipse area) at 1 and 3 months post-transplantation. (B): Representative image showing the distribution of CB-iNSCs in contralateral (left) hemisphere. (C): In vivo multilineage differentiation of CB-iNSCs at 1 month after transplantation. (D): Cell densities of migrated CB-iNSCs in contralateral (left) hemisphere at 1 and 3 months post-transplantation (p , .01). (E): Neurons differentiated from CB-iNSCs three months post-transplantation. Scale bars = 100 mm (A) and (B) and 50 mm (C) and (E). Abbreviations: DAPI, 49,6-diamidino-2phenylindole; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; mon., month.
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Furthermore, using a nonintegrative episomal vector, CB cells were converted into NSCs. The advantage of this latter approach is the elimination of a transgene in genomic DNA, as well as avoiding postconversion constitutive expression of a pluripotencyinducing transcription factor, OCT4. Indeed, as the converted cells are expanded and passaged, the episomal vector will get diluted by the time a cell therapy would be implemented. Although we did not observe tumorigenesis in iNSCs generated by the OCT4 lentivirus, the nonintegrative nature of the eiNSCs promises to be an even safer therapeutic strategy. Future efforts should focus on extending and optimizing in vitro culture of eiNSCs to be followed by animal studies.
CONCLUSION By directly converting CD34+ cord blood cells into neural stem cells, using only OCT4, we characterize a novel method of generating a patient-specific source of nontumorigenic, highly expandable neural tissue with a full spectrum of neurons, astrocytes, and oligodendrocytes. Our data are promising for future applications that require the safe generation of large amounts of autologous neural stem cells and neural tissue such as drug discovery, disease modeling, and regenerative medicine for neurological diseases.
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ACKNOWLEDGMENTS This work was partly supported by NIH Grant R01HL087948 (to Y.M.) and New York State Stem Cell Science Grants C028114 (to Y.M.), C026716 (to the Stony Brook Stem Cell Center), and 2013DFA30830 (to Y.J.).
AUTHOR CONTRIBUTIONS W.L.: conception and design, data analysis and interpretation, collection and/or assembly of data, manuscript writing; N.H. and J. Yu: collection and/or assembly of data, data analysis and interpretation; A.J., J. Yang, G.Z., X.J., and X.-B.Z.: data analysis and interpretation, manuscript writing; C.A: provision of study material; Y.M.: supervision of research, conception and design, data analysis and interpretation, manuscript writing.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST Y.M. is the founder of MarrowSource Therapeutics International LLC and an uncompensated patent holder. The other authors indicated no potential conflicts of interest. S TEM C ELLS T RANSLATIONAL M EDICINE
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Figure 5. Generation of eiNSCs with an episomal vector. (A): The diagram of the conversion procedure. (B): Adherent cell appearance after episomal transfection and in vitro culture of eiNSCs. (C): Nestin and MSI1 expression of eiNSCs. (D): The eiNSCs can differentiate into neurons and astrocytes as shown by Tuj1 and GFAP staining. Scale bars = 100 mm. Abbreviations: bFGF, basic fibroblast growth factor; DAPI, 49,6-diamidino-2phenylindole; EGF, epidermal growth factor; eiNSC, episomal vector-converted induced neural stem cell; GFAP, glial fibrillary acidic protein; hCB, human cord blood; MSI1, Musashi-1; SCF, stem cell factor; TPO, thrombopoietin.
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1 Gage FH. Mammalian neural stem cells. Science 2000;287:1433–1438. 2 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–676. 3 Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131:861–872. 4 Lujan E, Chanda S, Ahlenius H et al. Direct conversion of mouse fibroblasts to selfrenewing, tripotent neural precursor cells. Proc Natl Acad Sci USA 2012;109:2527–2532. 5 Meng X, Su RJ, Baylink DJ et al. Rapid and efficient reprogramming of human fetal and adult blood CD34+ cells into mesenchymal stem cells with a single factor. Cell Res 2013;23: 658–672. 6 Yu B, He ZY, You P et al. Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors. Cell Stem Cell 2013;13:328–340. 7 Yan Y, Shin S, Jha BS et al. Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:862–870. 8 WernigM,ZhaoJP,PruszakJetal.Neuronsderived from reprogrammed fibroblasts functionally
integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proc Natl Acad Sci USA 2008;105:5856– 5861. 9 Miura K, Okada Y, Aoi T et al. Variation in the safety of induced pluripotent stem cell lines. Nat Biotechnol 2009;27:743–745. 10 Han DW, Tapia N, Hermann A et al. Direct reprogramming of fibroblasts into neural stem cells by defined factors. Cell Stem Cell 2012; 10:465–472. 11 Ring KL, Tong LM, Balestra ME et al. Direct reprogramming of mouse and human fibroblasts into multipotent neural stem cells with a single factor. Cell Stem Cell 2012;11:100–109. 12 Sheng C, Zheng Q, Wu J et al. Direct reprogramming of Sertoli cells into multipotent neural stem cells by defined factors. Cell Res 2012;22:208–218. 13 Zangiacomi V, Balon N, Maddens S et al. Cord blood-derived neurons are originated from CD133+/CD34 stem/progenitor cells in a cell-to-cell contact dependent manner. Stem Cells Dev 2008;17:1005–1016. 14 Jurga M, Lipkowski AW, Lukomska B et al. Generation of functional neural artificial tissue from human umbilical cord blood stem cells. Tissue Eng Part C Methods 2009;15:365–372. 15 Jurga M, Forraz N, Basford C et al. Neurogenic properties and a clinical relevance of multipotent stem cells derived from cord blood
Tissue-Specific Progenitor and Stem Cells
TISSUE-SPECIFIC PROGENITOR AND STEM CELLS Direct Conversion of Cord Blood CD34+ Cells Into Neural Stem Cells by OCT4
Key Words. Direct conversion x Neural stem cell x Cord blood x OCT4 Departments of aPathology, b Surgery, and cObstetrics & Gynecology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York, USA; d Department of Medicine, Loma Linda University, Loma Linda, California, USA Correspondence: Wenbin Liao, M.D., Ph.D., Department of Pathology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York 11794, USA. Telephone: 949-824-7298; E-Mail: vwliao@ gmail.com; or Yupo Ma, M.D., Ph.D., Department of Pathology, Stony Brook University Hospital, Stony Brook University, Stony Brook, New York 11794, USA. Telephone: 631-444-2214; E-Mail: yupo.ma@ stonybrookmedicine.edu Received December 11, 2014; accepted for publication April 8, 2015. ©AlphaMed Press 1066-5099/2015/$20.00/0 http://dx.doi.org/ 10.5966/sctm.2014-0289
ABSTRACT Cellular reprogramming or conversion is a promising strategy to generate desired stem cell types from somatic cells. Neural stem cells (NSCs) have the potential to regenerate central nervous system tissue and repair damage in response to injury. However, NSCs are difficult to isolate from human tissues and expand in sufficient quantities for therapy. Here, we report a method to generate neural stem cells from cord blood CD34-positive cells by ectopic expression of OCT4 in a feeder-free system. The induced cells (iNSCs) show a characteristic NSC-like morphology and can be expanded in vitro for more than 20 passages. In addition, the iNSCs are positive for neural stem cell-specific markers such as Nestin and Musashi-1 and are similar in gene expression patterns to a human neural stem cell line. The iNSCs express distinct transcriptional factors for forebrain, hindbrain, and spinal cord regions. Upon differentiation, the iNSCs are able to commit into multilineage mature neural cells. Following in vivo introduction into NOD/SCID mice, iNSCs can survive and differentiate in the mouse brain 3 months post-transplantation. Alternatively, we were also able to derive iNSCs with an episomal vector expressing OCT4. Our results suggest a novel, efficient approach to generate neural precursor cells that can be potentially used in drug discovery or regenerative medicine for neurological disease and injury. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:1–9
SIGNIFICANCE This study describes a novel method to generate expandable induced neural stem cells from human cord blood cells in a feeder-free system by a single factor, OCT4. The data are promising for future applications that require the generation of large amounts of autologous neural stem cells in disease modeling and regenerative medicine.
INTRODUCTION Neural stem cells (NSCs) can generate the vast array of neurons, astrocytes, and oligodendrocytes that comprise the nervous system. Consequently, NSCs hold significant potential as a therapeutic strategy for treatment of spinal cord injury, as well as neurodegenerative diseases such as Parkinson’s disease [1]. In the past, the use of NSCs for the treatment of neurological disease was hindered by limited fetal and adult NSC sources. Recent advances in cellular reprogramming allowed for the generation of induced pluripotent stem cells (iPS) and somatic stem cells from various somatic tissues [2–6]. To that effect, NSCs can be obtained by differentiation from iPS cells [7, 8]. However, differentiating iPS cells into NSCs presents with numerous drawbacks that need to be addressed before clinical applications: prolonged culture time, putative low reprogramming efficiency, and tumorigenic risk because of the possible presence of residual pluripotent cells
postdifferentiation [9]. An alternative and safer strategy may be to bypass the pluripotent state and convert directly to NSCs. Indeed, several groups have described successful direct conversion from fibroblasts or other somatic cells to NSCs [4, 10–12]. However, the generation of NSCs required the use of feeder cells, a potential limitation for any cell therapy-based application. In our study, we describe the generation of expandable NSCs from human cord blood CD34-positive cells using a novel method that is efficient, does not require feeder cells, and is mediated by a single factor, OCT4. We also describe a nonintegrating, episomal vector for OCT4-mediated generation of induced NSCs from cord blood.
MATERIALS AND METHODS Cell Culture Cord blood (CB) CD34+ cells were isolated using human CD34+ labeling kits (Miltenyi Biotec,
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WENBIN LIAO,a NICK HUANG,a JINGXIA YU,a ALEXANDER JARES,a JIANCHANG YANG,b GARY ZIEVE,a CECILIA AVILA,c XUN JIANG,a XIAO-BING ZHANG,d YUPO MAa
Published Ahead of Print on May 13, 2015 as 10.5966/sctm.2014-0289.
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Direct Conversion of CD34+ Cells Into NSCs by OCT4 Lentivirus and Episomal Vector Lentiviruses carrying human OCT4 gene or control green fluorescent protein (GFP) gene under spleen focus-forming virus (SFFV) promoter were packaged and produced using the 293FT cell line. The viruses were concentrated by centrifugation and stored at 280°C. Fresh isolated or frozen human CD34+ cells were cultured in growth medium for 24 hours before transduction. Lentiviruses were added at a multiplicity of infection of 2–5 in the presence of 8 mg/ml Polybrene (Millipore) for 4–6 hours (day 0). Then transduced cells were washed in warm phosphatebuffered saline (PBS) and seeded at a density of ∼20,000 cells per mm2 in tissue culture plates that were precoated with 10 mg/ml fibronectin. The next day (day 1), half of the growth medium was removed, and the same amount of Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (DMEM/DF12 supplemented with 15% FBS, 10 ng/ml human bFGF, and 100 U/ml penicillin/streptomycin) was added. On day 2, the medium was changed to DMEM/F12 medium completely and refreshed every 2 days. On day 7, the adherent cells reached confluence and were replated at a density of 50,000 cells per mm2 after Accutase (Millipore) digestion. When the cells were confluent again, all cells were dissociated into single cells and plated at a density of 50,000 cells per mm2 on laminin-coated (20 mg/ml) plates in ReNcell medium. Routinely, the induced cells (CB-iNSCs) were passaged every 3–4 days with a doubling time of ∼24 hours. For neurosphere formation, the CBiNSCs were seeded in low attachment Petri dishes at a density of 200,000 cells per ml in ReNcell medium. During the cellular conversion procedure, 3 mM CHIR99021 (Stemgent, Cambridge, MA, https://www.stemgent.com) was used starting from the time of lentiviral transduction until the second passage in ReNcell medium. ReNcell medium was changed every 2–3 days. For conversion of CD34+ cells to iNSCs by OCT4 episomal vector (eiNSCs), the CD34+ cells were transfected with EBNV1based pCEP4-OCT4 expression plasmid by electroporation using a human CD34 cell nucleofector kit (Lonza, Koln, Germany, http://www.lonza.com). Cell culture and media changes after transfection were similar to those for the lentivirus-based cellular conversion, except for the fact that hypoxic conditions (5% O2) were required to generate eiNSCs. 3-Deazaneplanocin (Dzep; Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich. com) was added to the solution after the first passage in ReNcell medium.
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Differentiation of CB-iNSCs For induction of CB-iNSCs to three neural cell lineages, CB-iNSCs were cultured in poly-L-ornithine/laminin-coated glass coverslips in ReNcell medium without bFGF and EGF for 14 days, which allowed random differentiation and maturation of CB-iNSCs. For specific differentiation into neural cells, cells were induced by addition of 5 mM forskolin (Sigma), 20 ng/ml brain-derived neurotrophic factor, and 20 ng/ml glial cell-derived neurotrophic factor (GDNF; PeproTech, Rocky Hill, NJ, https://www.peprotech.com). For oligodendrocyte differentiation, CB-iNSCs were stimulated with 100 ng/ml Sonic hedgehog (SHH) for 4 days and then cultured in DMEM/F12 supplemented with 2% B27, 10 ng/ml platelet-derived growth factor AA (Peprotech), and 40 ng/ml thyroid hormone T3 (Sigma) for 3 weeks with half medium change twice each week. For dopaminergic neuron differentiation, CB-iNSCs were induced by human 100 ng/ml FGF-8 and 100 ng/ml SHH for 3 days and by 20 ng/ml ciliary neurotrophic factor and 25 ng/ml GDNF for 2 weeks.
Electrophysiology Whole-cell patch clamp was performed to measure the membrane current in single cells from undifferentiated or neurally differentiated CB-iNSCs or hcx NSCs. The cells were replated on polylysine-coated coverslips 1 day before the assay. The patch pipette resistances were 1–3 MV before sealing. The pipette solution contained (in mmol/l) 50 KCl, 80 potassium aspartate, 1 MgCl2, 3 Mg-ATP, 10 EGTA, and 10 HEPES (pH adjusted to 7.2 with KOH). The extracellular solution contained (in mmol/l) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH adjusted to 7.4 with NaOH). Voltage and current signals were recorded by the amplifiers (model Axopatch-1B; Axon Instruments/Molecular Devices Corp., Union City, CA, http://www.moleculardevices.com), digitized (model DIGIDATA 1320A digitizers; Axon Instruments/ Molecular Devices Corp.), and finally transferred to a personal computer.
Regular PCR and Quantitative PCR Total RNA was extracted with an AllPrep DNA/RNA mini kit (Qiagen, Hilden, Germany, http://www.qiagen.com), and cDNA was synthesized using a QuantiTect reverse transcription kit (Qiagen). Polymerase chain reaction (PCR) amplification was conducted with Platinum PCR Supermix, high fidelity (Life Technologies, Grand Island, NY, http://www.lifetechnologies.com). Quantitative PCR (qPCR) was run on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) with Power SYBR Green PCR Master Mix (Life Technologies). All qPCRs were conducted in triplicate. The expression results of real-time PCR were presented by log2fold according to D(DCt), which is normalized to b-actin expression. The detailed information for all primers is provided in supplemental online Table 2.
Gene Expression Microarray Total RNAs of CB CD34+ cells, CB-iNSCs, and hcx NSCs were extracted using the kits as mentioned above. RNA quantity and quality (2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA, http://www.agilent.com) was determined to be optimal before further processing. The Affymetrix (Santa Clara, CA, http://www. affymetrix.com) human HG-U133plus2 GeneChip arrays hybridization, staining, and scanning were performed using Affymetrix standard protocols as previously described by the Stony Brook S TEM C ELLS T RANSLATIONAL M EDICINE
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Auburn, CA, http://www.miltenyibiotec.com) and cultured in Stemspan medium (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) supplemented with 10% fetal bovine serum (FBS), 100 ng/ml human stem cell factor, 100 ng/ml human thrombopoietin, 100 ng/ml human fms-like tyrosine kinase 3 ligand (Peprotech, Rocky Hill, NJ, http://www.peprotech.com), and 100 U/ml penicillin/streptomycin (Gibco, Grand Island, NY, http://www. invitrogen.com). A human cortex (hcx) neural progenitor cell line was purchased from Millipore (Billerica, MA, http://www. millipore.com) and maintained in ReNcell medium: ReNcell NSC maintenance medium (Millipore) supplemented with 20 ng/ml human epidermal growth factor (EGF), 20 ng/ml human basic fibroblast growth factor (bFGF) (Peprotech), and 100 U/ml penicillin/ streptomycin.
Inducing Neural Stem Cells From Cord Blood by OCT4
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University DNA Microarray Core Facility. All genes of neurogenesis and hematopoiesis according to Gene Ontology terms (AmiGO, http://www.geneontology.org) were analyzed, and upregulation or downregulation fold changes were normalized to CB CD34+ cells. The heat map of gene expression levels was generated by R software.
Transplantation of CB-iNSCs Into Mice
Immunostaining Cultured cells or brain sections were fixed in 4% paraformaldehyde for 15 minutes at room temperature and washed with PBS. Nonspecific antibody binding was blocked by using 1% bovine serum albumin for 30 minutes, and cells were permeabilized with 0.3% Triton X-100 (Sigma) in PBS (PBS-T) for 30 minutes at room temperature. Cells were rinsed and then incubated in primary antibody containing 0.1% overnight at 4°C. After washing in PBS, the cells were incubated in secondary antibody for 1 hour at room temperature. The cells were immunostained with the following anti-human primary antibodies: anti-Nestin, anti-Musashi1, anti-tyrosine hydroxylase (TH), anti-bIII tubulin (Tuj1), anti-glial fibrillary acidic protein (GFAP), and anti-29,39-cyclic nucleotide 39-phosphodiesterase (CNPase). Primary antibodies were detected with the phosphatidylethanolamine- or fluorescein isothiocyanate-conjugated secondary antibody. 49,6-Diamidino-2phenylindole (DAPI) was used to identify cell nuclei. Stained cells were preserved in antifading mount solution that contained DAPI. Stained cells were examined and photographed under an EOVS fluorescent microscope (Life Technolgies).
Statistical Analysis The results are presented as mean 6 SEM. Two-tailed Student’s t test was performed for comparison. p , .01 was considered statistically significant.
RESULTS Conversion of Cord Blood CD34+ Cells Into Neural Stem Cells in a Feeder-Free System Previously, human cord blood cells were reported to contain cells with neurogenic properties [13–15]. In order to detect whether
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there are any neural precursor cells in the CD34+ fraction of CB, we cultured CB CD34+ cells directly on laminin-coated tissue culture plates in ReNcell medium in the presence of human EGF and bFGF, which is a well established and standard culture condition for human neural stem cells (Millipore). In repeated attempts, we did not obtain any adherent cells resembling NSCs. In addition, we failed to detect the expression of any of the well-known neural stem cell genes in CB CD34+ cells by PCR (supplemental online Fig. 1B). Flow cytometry analysis further confirmed the absence of Nestin in the CB CD34+ cells (supplemental online Fig. 1C). These results suggest the absence of neural precursor cells in the original CD34+ cell population. We transduced cord blood CD34+ cells with lentiviruses carrying OCT4 or control GFP gene driven by an SFFV promoter. After OCT4 transduction in human cord blood CD34+ cells, adherent spindle-like cells could be observed as early as 24 hours posttransduction. When cultured in medium containing human bFGF, the derived adherent cells exhibited high proliferation potency (Fig. 1). In contrast, in the control GFP vector transduction, although a few adherent cells were detected in culture posttransduction, none displayed proliferative properties. Additionally, after multiple attempts with transduction with OCT4, cord blood CD342 cells did not generate proliferating adherent cells. In our system, the adherent cells in OCT4 transduced CD34+ CB groups consistently reached confluence 6–8 days after transduction, at which point, the number of adherent cells was ∼10-fold the number of the initial CD34+ cells. Consequently, at passage 1, we obtained ∼100-fold the number of induced NSCs (CB-iNSCs) relative to the initial CD34+ cell number (supplemental online Table 1). These CB-iNSCs were highly expandable (.20 passages, .2 months in culture) with unchanged morphology and stable doubling times (24–36 hours) in vitro (Fig. 1). In order to determine whether iPS cells were initially derived and subsequently programmed to neural stem cells in the culture, we stained the adherent cells at different time points (days 3 and 6 after OCT4 transduction) with TRA-1-60, TRA-1-81, and SSEA-3/4; none of these surface markers positively reacted with the cells (data not shown). In addition, we did not see teratoma formation after subcutaneous injection of CB-iNSCs in NOD/SCID mice (supplemental online Table 3). Thus, the data suggested that iNSCs were directly converted from CD34+ cells without a pluripotent stem cell intermediate step.
Characterization of CB-iNSCs The CB-iNSCs presented with characteristic NSC-like morphology. Subsequent immunostaining revealed that CB-iNSCs expressed neural stem cell markers such as Nestin, Musashi-1 (MSI1), and Pax6 (Fig. 2A). We also quantified the gene expression levels of CB-iNSCs by real-time PCR (Fig. 2B). As compared with the original cord blood cells, the neural stem cell markers Nestin, MSI1, Sox1, and Pax6 were significantly upregulated, whereas the hematological markers CD34, CD38, and CD45 were markedly downregulated. The gene expression patterns of neural stem cells and hematopoietic markers are similar between CB-iNSCs and hcx NSCs as compared with CB cells. Of note is that the overall gene expression levels were elevated in hcx NSCs for both neural stem cells and hematopoietic markers. This may be due to the ectopic expression of oncogene c-MYC in the hcx NSCs line. Further analysis using gene expression arrays confirm that CB-iNSCs are similar to hcx NSCs in gene expression patterns in neurogenesis and hematopoiesis (1578 genes in total) (Fig. 2C; supplemental online Fig. 2; supplemental online Tables 4, 5). We also compared the
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To track the CB-iNSCs in vivo, CB-iNSCs were labeled by transduction of GFP lentivirus before injection to the right striatum of NOD/SCID mice. Under anesthesia, the right side of the mouse skulls were exposed after tissue separation. A hole with a diameter of approximately 1.0 mm was drilled at center of the coordinates: anterior-posterior (AP): 0 mm; medial-lateral (ML): 2.5 mm. Then NOD/SCID mice were placed under stereotaxic apparatus and received 200,000 CB-iNSCs (in 2 ml of PBS) at the coordinates: AP: 0 mm; ML: 2.5 mm; dorsal-ventral: 3.5 mm. The cell injection was finished in 5 minutes, and the syringe was removed after another 5 minutes. Antibiotics (0.5% Bactrim in water) were administered postsurgically to the animals for 2 weeks. The cryostat sections (10 mm) of the animals’ brains were prepared after intracardiac perfusion, fixation, and dehydration 1 or 3 months after transplantation. The density of migrated cells in the contralateral hemisphere (left brain) was analyzed by counting GFP+ cells in six random fields of the coronal sections (AP: 0 mm) under a 310 lens of the fluorescent microscope.
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gene expression pattern of early and late passages CB-iNSCs and found no obvious differences between the results of passage 3 and passage 10 CB-iNSCs (Fig. 2B), suggesting maintained stem cell signature even during prolonged in vitro culture. Additionally, CB-iNSCs could form neurospheres in low-adherent culture dishes (Fig. 2D). Therefore, CB-iNSC gene expression profiles and morphology are highly consistent with neural stem cells.
Differentiation of CB-iNSCs In Vitro We induced the differentiation of the CB-iNSCs by simple removal of growth factors using a series of cytokines and chemicals. When the growth factors (EGF and bFGF) were removed from the ReNcell medium, the growth speed of CB-iNSCs largely slowed down, and the cells underwent differentiation into more long neurites, which increased in number and further matured with additional culture time (Fig. 3A; supplemental online Fig. 3). Synapse-like structures between cells could be seen after 2 weeks of in vitro differentiation. This is consistent with positive synapsin1 expression detected by PCR assay (Fig. 3C, 3D). By immunostaining, we found expression of Tuj1, GFAP, or CNPase in the differentiated cells, indicating the multilineage differentiation capacity of CBiNSCs (Fig. 3B). This was also confirmed by PCR results, in which markers of all three lineages of neural cells were detected: Tuj1, MAP2, and neurofilament (NF) for neurons; S100B for astrocytes and MBP; and CNPase for oligodendrocytes (Fig. 3D). In addition, we saw expression of both vesicular glutamate transporter 1 (VGLUT1) and g-aminobutyric acid (GABA) receptor in the differentiated cells, suggesting that CB-iNSCs could generate both excitatory (glutamatergic) and inhibitory (GABAergic) neurons. In an electrophysiological assay, we found that patterns of sustained potassium currents in response to stepwise voltage stimulations were similar between CB-iNSCs and hcx NSC-derived neural cells,
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suggesting similar functional properties across these two cell types (Fig. 3G).
Regional Pattern of CB-iNSCs In the development of the neural system, there are distinct expressions of transcriptional factors in the precursors for forebrain, midbrain and hindbrain regions. We detected forebrain (FOXG1), hindbrain (HOXA2, GBX1, EGR2, and HOXB2), and also spinal cord (HOXB6) markers (Fig. 3E), but not midbrain markers (PAX2 and EN1) in our CB-iNSCs (data not shown). Interestingly, we observed the same gene expression profile in passage 3 and passage 10 CB-iNSCs, suggesting that forebrain and hindbrain region-specific markers did not change over time (Fig. 3E). Control hcx NSC expressed forebrain (FOXG1) and hindbrain (GBX1, EGR2 and HOXB2) markers (Fig. 3E) and did not express midbrain markers (data not shown). The original CB CD34+ cells did not express any of the all midbrain and forebrain markers tested and were negative for hindbrain GBX1 as well. Notably, CB cells are positive for EGR2, HOXA2, HOXB2, and HOXB6 makers because they are also related to adult hematopoiesis [16, 17]. To check whether the CB-iNSCs could be induced for midbrain neuron commitment, we induced these CB-iNSCs in specific medium condition for midbrain dopaminergic neuron differentiation. The results showed that TH+/Tuj1+ positive cells could be detected after 2 weeks of induction (Fig. 3F). These data demonstrate that CB-iNSCs are readily expressing genes of forebrain, hindbrain, and spinal cord regions and can also be patterned to midbrain fate after specific stimuli.
CB-iNSCs Engraftment in Mouse Brain We labeled the CB-iNSCs with GFP marker and injected them into NOD/SCID mouse striatum under stereotaxic surgery. We found S TEM C ELLS T RANSLATIONAL M EDICINE
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Figure 1. Conversion of CB CD34+ cells to iNSCs by lentiviral OCT4 overexpression. (A): Diagram of the cellular conversion procedure. (B): Adherent appearance in OCT4 and control group. (C): In vitro long-term expansion of CB-iNSCs. (D): Growth curve of CB-iNSCs (n = 4). Scale bars = 50 mm. Abbreviations: bFGF, basic fibroblast growth factor; CB, cord blood; EGF, epidermal growth factor; hCB, human cord blood; iNSC, induced neural stem cell; Pn, passage n; SCF, stem cell factor; TPO, thrombopoietin.
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that a large proportion of these cells still survived in the brain 1 month after transplant (Fig. 4A). Long neurites could be detected from some of the cells. By immunostaining, we found Tuj1-, GFAP-, or CNPase-expressing cells from the GFP-positive injected cells, suggesting the in vivo maturation of CB-iNSCs (Fig. 4C). The injected cells are still observed 3 months post-transplantation (Fig. 4A). The cell number at the injection sites at 3 months is less than at 1 month post-transplantation (Fig. 4A); however, we found more cells in regions other than the injection site
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(Fig. 4B, 4D; supplemental online Fig. 4). We also detected Tuj1 expression of the injected cells 3 months post-transplantation (Fig. 4E). These data suggest that CB-iNSCs can survive, mature, and migrate long term in an in vivo setting. We also monitored the mice after receiving CB-iNSCs for long-term safety, and no abnormalities were observed more than 3 months postinjection (supplemental online Table 3). Therefore, CB-iNSCs are able to engraft and differentiate in the central nervous system without tumor formation.
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Figure 2. Characterization of CB-iNSCs. (A): Nestin, MSI1, PAX6 expression of CB-iNSC1, CB-iNSC3, and hcx NSC. (B): Relative expression of neural stem cells or hematopoietic stem cell genes of CB-iNSCs to CB CD34+ cells as quantitated by real-time polymerase chain reaction analysis. (C): Heat map of expression of genes in neurogenesis and hematopoiesis (a total of 1,578 genes) of CB-iNSCs and hcx NSCs by gene expression microarray. The expression levels of CB-iNSCs and hcx NSCs were presented in Log2fold change after being normalized to CB cells. (D): Neurosphere formation of CB-iNSCs. Scale bars = 50 mm. Abbreviations: CB, cord blood; DAPI, 49,6-diamidino-2-phenylindole; hcx, human cortex; iNSC, induced neural stem cell; MSI1, Musashi-1; NSC, neural stem cell; Pn, passage n.
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Inducing Neural Stem Cells From Cord Blood by OCT4
Generation of Integration-Free NSCs With an Episomal Vector Although direct conversion using lentivirus does provide an increasingly popular approach that bypasses the iPS cell state, there is the risk that random integration of foreign DNA into the host’s genome would potentially lead to insertional mutagenesis. To that effect, we sought to generate iNSCs from CB using an integration-free method. CB CD34+ cells were electroporated with an episomal vector containing a high expression OCT4 gene (Fig. 5A). After nucleofection, we successfully generated neural stem cells within 2–3 weeks (Fig. 5B), which we hereafter refer to as eiNSCs for brevity. iNSCs and eiNSCs are identical morphologically, and eiNSCs did not differ in the expression of typical NSC markers of Nestin and Musashi (Fig. 5C). In addition, eiNSCs are able to differentiate into neural and glial cells because neuron and astrocyte markers were observed after random differentiation (Fig. 5D). However, eiNSCs are slower in growth rate with a doubling time of approximately 36 hours, whereas iNSCs have a doubling time of approximately 24 hours. Also, eiNSCs failed to maintain long-term expansion in vitro because no growth was observed after three to four passages. When Dzep, a compound that was recently reported to increase the efficiency of iPS generation [18], was added to the culture, eiNSCs grew for two additional passages (Fig. 5B).
DISCUSSION OCT4 is a key transcriptional factor in the reprogramming of somatic cells to iPS cells [2, 3]. Our findings establish that the
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introduction of OCT4 alone is enough to directly convert mesoderm-originated cells (human cord blood derived CD34+ cells) to neuroectoderm cells without a pluripotent intermediate. Our method is a rapid and efficient approach to obtain highly expandable iNSCs that are able to differentiate into all three neural lineage cells. Given that the initiating cord blood cells grow as a suspension culture and that induced NSCs grow as adherent tissue culture cells, cellular conversion events are easy to detect and track. Furthermore, the iNSC adherent cells proliferate rapidly during cellular conversion. By passage 1, that is, within 2 weeks of conversion, we obtained an iNSC number that is a 100-fold increase relative to the starting CD34+ population. Crucially for therapeutic applications, the iNSCs are consistently able to maintain stable long-term expansion in vitro. Therefore, a single cord blood sample can yield a large amount of autologous iNSCs for use in drug screenings or regenerative cell therapy. The OCT4 expression level determines in part whether pluripotent stem cells maintain stemness or head to differentiation. In our study, sufficient Oct4 expression level is critical for successful CB-iNSCs generation. For instance, when Oct4 is driven by a CMV promoter, OCT4 expression is 50% lower than for the SFFV-Oct4 vector. As a result, lentiviral transduction of OCT4 driven by CMV promoter is not sufficient to convert CB cells into iNSCs. This is especially salient in a single-factor cellular reprogramming or conversion system. In previous reports, iNSCs were generated from a variety of cell sources [19–22]. However, translation into cell therapy applications, for which large amounts of cells are needed, was hindered by the limited proliferative potential of the derived iNSCs. Subsequent attempts by several groups generated more S TEM C ELLS T RANSLATIONAL M EDICINE
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Figure 3. In vitro differentiation of CB-iNSCs. (A): Differentiated CB-iNSCs show neuron-like morphology with long neurites as compared with undifferentiated cells. (B): Multilineage differentiation of CB-iNSCs as shown by staining of neuron (Tuj1), astrocyte (GFAP), or oligodendrocyte (CNPase) marker immunostaining. (C): Synapse-like structure in differentiated CB-iNSCs. (D): Polymerase chain reaction analysis of genes related to neurotransmitter and multilineage neural cells. i: Undifferentiated CB-iNSC 1. ii: Differentiated CB-iNSC 1. iii: Differentiated CB-iNSC 3. (E): Regional pattern of CB-iNSCs. (F): Dopaminergic neural cell differentiation from CB-iNSCs. (G): Representative voltage-clamp recordings of potassium channel currents in response to stepwise voltage stimulations (2120 mV to +120 mV) for neurally differentiated cells from CB-iNSCs and control hcx NSCs. Scale bars = 50 mm. Abbreviations: CB, cord blood; DAPI, 49,6-diamidino-2-phenylindole; GABA, g-aminobutyric acid; GFAP, glial fibrillary acidic protein; hcx, human cortex; iNSC, induced neural stem cell; NF, neurofilament.
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expandable iNSCs using combinatorial expression of a set of transcriptional factors [4, 10, 12]; however, obstacles to cell therapy remained. Those iNSCs retained restricted differentiation potential and the use of oncogenic factors, such as c-MYC, highlighted the risks of long-term constitutive expression of reprogramming transgenes after the target tissue had already been derived. Indeed, a recent study reported the generation of iNSCs from cord blood CD45+/CD133+ cells with SOX2 alone and with the addition of c-MYC. Another separate study reported the generation of iNSCs from fibroblasts using SOX2 alone [11, 23]. In the first study [23], the iNSCs from cord blood cells showed restricted neural lineages immediately after conversion, and prolonged cell culture in permissive conditions is required to expand and further dedifferentiate the induced neuronal cells to an earlier cell stage. In the second study [11], SOX2 was used to convert mouse or human fibroblasts into neural precursor cells; however, there remain no in vivo data on human iNSCs. Additionally, in both of these studies, feeder cells were required for conversion. In contrast, we were able to consistently generate CB-iNSCs without support from a feeder
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cell system. We also attempted to convert human cord blood CD342 cells or human skin fibroblasts into NSCs with the feeder-free system; however, no iNSCs were successfully obtained (data not shown). Thus, CD34+ cells are an easily obtainable and efficient source for patient-specific NSC conversion. CB-iNSCs are able to give rise to multilineage neural cells both in vitro and in vivo. Similar to NSCs isolated from neural tissues, CB-iNSC expansion is dependent on stimulation with human EGF and bFGF and the removal of growth factors that lead to cell maturation. Intriguingly, CB-iNSCs show multiple regional patterns including forebrain, hindbrain, and spinal cord, and these patterns are maintained at least for 10 passages. We also found that FGF-8/SHH stimulation could induce the generation of midbrain-specific cells. Importantly for long-term translational applications, CB-iNSC consistently maintained differentiation capacity in vivo. In addition, the CB-iNSCs are able to survive at least 3 months without any aberrant proliferation in the mouse brain, suggesting long-term engraftment and safety in vivo.
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Figure 4. In vivo transplantation of cord blood (CB)-induced neural stem cells (iNSCs). (A): The GFP-labeled CB-iNSCs (green) are visible in the injection region (right striatum, marked ellipse area) at 1 and 3 months post-transplantation. (B): Representative image showing the distribution of CB-iNSCs in contralateral (left) hemisphere. (C): In vivo multilineage differentiation of CB-iNSCs at 1 month after transplantation. (D): Cell densities of migrated CB-iNSCs in contralateral (left) hemisphere at 1 and 3 months post-transplantation (p , .01). (E): Neurons differentiated from CB-iNSCs three months post-transplantation. Scale bars = 100 mm (A) and (B) and 50 mm (C) and (E). Abbreviations: DAPI, 49,6-diamidino-2phenylindole; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; mon., month.
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Inducing Neural Stem Cells From Cord Blood by OCT4
Furthermore, using a nonintegrative episomal vector, CB cells were converted into NSCs. The advantage of this latter approach is the elimination of a transgene in genomic DNA, as well as avoiding postconversion constitutive expression of a pluripotencyinducing transcription factor, OCT4. Indeed, as the converted cells are expanded and passaged, the episomal vector will get diluted by the time a cell therapy would be implemented. Although we did not observe tumorigenesis in iNSCs generated by the OCT4 lentivirus, the nonintegrative nature of the eiNSCs promises to be an even safer therapeutic strategy. Future efforts should focus on extending and optimizing in vitro culture of eiNSCs to be followed by animal studies.
CONCLUSION By directly converting CD34+ cord blood cells into neural stem cells, using only OCT4, we characterize a novel method of generating a patient-specific source of nontumorigenic, highly expandable neural tissue with a full spectrum of neurons, astrocytes, and oligodendrocytes. Our data are promising for future applications that require the safe generation of large amounts of autologous neural stem cells and neural tissue such as drug discovery, disease modeling, and regenerative medicine for neurological diseases.
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ACKNOWLEDGMENTS This work was partly supported by NIH Grant R01HL087948 (to Y.M.) and New York State Stem Cell Science Grants C028114 (to Y.M.), C026716 (to the Stony Brook Stem Cell Center), and 2013DFA30830 (to Y.J.).
AUTHOR CONTRIBUTIONS W.L.: conception and design, data analysis and interpretation, collection and/or assembly of data, manuscript writing; N.H. and J. Yu: collection and/or assembly of data, data analysis and interpretation; A.J., J. Yang, G.Z., X.J., and X.-B.Z.: data analysis and interpretation, manuscript writing; C.A: provision of study material; Y.M.: supervision of research, conception and design, data analysis and interpretation, manuscript writing.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST Y.M. is the founder of MarrowSource Therapeutics International LLC and an uncompensated patent holder. The other authors indicated no potential conflicts of interest. S TEM C ELLS T RANSLATIONAL M EDICINE
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Figure 5. Generation of eiNSCs with an episomal vector. (A): The diagram of the conversion procedure. (B): Adherent cell appearance after episomal transfection and in vitro culture of eiNSCs. (C): Nestin and MSI1 expression of eiNSCs. (D): The eiNSCs can differentiate into neurons and astrocytes as shown by Tuj1 and GFAP staining. Scale bars = 100 mm. Abbreviations: bFGF, basic fibroblast growth factor; DAPI, 49,6-diamidino-2phenylindole; EGF, epidermal growth factor; eiNSC, episomal vector-converted induced neural stem cell; GFAP, glial fibrillary acidic protein; hCB, human cord blood; MSI1, Musashi-1; SCF, stem cell factor; TPO, thrombopoietin.
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