EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS Neural Stem Cells Directly Differentiated from Partially Reprogrammed Fibroblasts Rapidly Acquire Gliogenic Competency TAKESHI MATSUI,a MORITO TAKANO,b KENJI YOSHIDA,a SOICHIRO ONO,a CHIKAKO FUJISAKI,a YUMI MATSUZAKI,a YOSHIAKI TOYAMA,b MASAYA NAKAMURA,b HIDEYUKI OKANO,a WADO AKAMATSUa Department of Physiology and bDepartment of Orthopedic Surgery, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, Japan a
Key Words. Direct cell conversion • Neural stem cell • Reprogramming • Neural induction • Induced pluripotent stem cells
ABSTRACT Neural stem cells (NSCs) were directly induced from mouse fibroblasts using four reprogramming factors (Oct4, Sox2, Klf4, and cMyc) without the clonal isolation of induced pluripotent stem cells (iPSCs). These NSCs gave rise to both neurons and glial cells even at early passages, while early NSCs derived from clonal embryonic stem cells (ESCs)/ iPSCs differentiated mainly into neurons. Epidermal growth factor-dependent neurosphere cultivation efficiently propagated these gliogenic NSCs and eliminated residual pluripotent cells that could form teratomas in vivo. We con-
cluded that these directly induced NSCs were derived from partially reprogrammed cells, because dissociated ESCs/ iPSCs did not form neurospheres in this culture condition. These NSCs differentiated into both neurons and glial cells in vivo after being transplanted intracranially into mouse striatum. NSCs could also be directly induced from adult human fibroblasts. The direct differentiation of partially reprogrammed cells may be useful for rapidly preparing NSCs with a strongly reduced propensity for tumorigenesis. STEM CELLS 2012;30:1109–1119
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION The direct reprogramming of somatic cells into other cell lineages facilitates the rapid and efficient production of target cells, which may be used for cell therapies in the future. By introducing tissue-specific transcription factors, neurons [1, 2], cardiomyocytes [3], and cartilage [4] can be generated from skin fibroblasts. However, these transdifferentiated cells have a limited ability to proliferate and differentiate into multiple progenitors. In contrast, partially reprogrammed cells induced using Yamanaka factors (Oct4, Klf4, Sox2, and c-Myc) [5, 6] or Oct4 alone [7] can generate tissue-specific stem/progenitor cells under the appropriate culture conditions. Although reports claim that these cells are not generated from induced pluripotent stem cells (iPSCs) but from intermediate metastable states, it is difficult to exclude the possibility that some of these cells transiently go through a pluripotent stage during the reprogramming and differentiation processes. Among iPS-derived tissue stem/progenitor cells, residual pluripotent cells that are resistant to forced differentiation give rise to teratomas with three germ layers [8]. We previously showed that safe mouse iPS clones must be selected to achieve efficient functional recovery without tumor formation in spinal cord
injury (SCI) model mice by iPS-derived neural stem cell (NSC) transplantation [9]. However, we also showed that more than 80% of iPS clone-derived NSCs generate detectable teratomas after intracranial injection, when the iPS clones are established from adult skin fibroblasts [8]. ‘‘Safe’’ iPS clones must be carefully selected to prevent teratoma formation. In the differentiation of embryonic stem cells (ESCs)/iPSCs into NSCs, neurogenic NSCs appear first, and they develop into gliogenic NSCs during the recurrent passages in vitro [10]. Such gliogenic NSCs are effective for treating SCI model animals, but neurogenic NSCs are not [9, 11]. For NSCs transplantation to be effective in SCI patients, it must be performed before the chronic phase [12]; therefore, the rapid induction of gliogenic NSCs with reduced tumorigenicity will be required for future treatments involving autograft transplantation. In this report, we introduce a novel culture system that generates NSCs from adult mouse fibroblasts that were partially reprogrammed by introducing the four Yamanaka factors, and that directs their differentiation into neural lineages. Interestingly, the NSCs derived by this method differentiated into both neuronal and glial cells, even at early passages. We suggest that these gliogenic NSCs, which differentiate more rapidly than ESC/iPSC-derived NSCs, were derived from partially reprogrammed cells, because dissociated ESCs/iPSCs
Author contributions: T.M. and K.Y.: collection and assembly of data, data analysis and interpretation, and manuscript writing; M.T., C.F., S.O., and Y.M.: collection and assembly of data; Y.T.: conception and design; M.N.: conception and design and financial support; H.O. and W.A: conception and design, financial support, administrative support, manuscript writing, and final approval of manuscript. H.O. and W.A. contributed equally to this article. Correspondence: Wado Akamatsu, M.D., Ph.D., Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Telephone: þ81-3-5363-3747; Fax: þ81-3-3357-5445; e-mail:
[email protected]; or Hideyuki Okano, M.D., Ph.D., Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Telephone: þ81-3-5363-3747; Fax: þ81-3-3357-5445; e-mail:
[email protected] Received August 23, 2011; accepted for C AlphaMed Press 1066-5099/2012/$30.00/0 publication March 1, 2012; first published online in STEM CELLS EXPRESS March 29, 2012. V doi: 10.1002/stem.1091
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did not form neurospheres under the same culture conditions. Furthermore, we induced human NSCs directly from adult fibroblasts using this method.
MATERIALS
AND
METHODS
Cell Culture Adult skin fibroblasts were obtained from 8-week-old C57BL/ 6J mice. To obtain these cells, the skin was peeled from the body of adult mice, minced into 5-mm pieces, placed on culture dishes, and incubated in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum (FBS), 50 U penicillin, and 50 mg/ml streptomycin. Cells that migrated out of the skin pieces were trypsinized and transferred to new plates. We used the adult mouse fibroblasts at passage 3-5 for direct neural induction.
culture period, the fibroblasts were filmed for 14 days continuously in the BioStation CT [16].
Bisulfite PCR The genomic DNA was extracted with a Qiagen DNeasy kit (Qiagen, Venlo, Netherlands, www.qiagen.com/). The purified genomic DNA was denatured and converted with a Qiagen Epitect kit. The bisulfite-modified DNA was purified and used as a template for polymerase chain reaction (PCR). The PCR products were subcloned into PT7blue (Novagen, Darmstadt, Germany, www.merckgroup.com/en/index.html), and individual clones were randomly selected for DNA sequencing with U19 primers for each gene. The PCR primers are listed in Supporting Information Table S1.
Immunocytochemical and Immunohistochemical Analyses
Retroviral infection was performed as described previously [13]. Briefly, the day before transfection, Plat-E cells were seeded at 3.6 106 cells per 10-cm dish. The next day, pMXbased retroviral vectors were introduced into the Plat-E cells by the Fugene 6 transfection reagent (Roche, Penzberg, Germany, www.roche.com/index.htm). The virus-containing supernatant was used to infect the fibroblasts, seeded at 8 105 cells per 10-cm gelatin-coated dish. The infected cells were collected and subjected to neural induction by suspension culture.
Immunocytochemical and immunohistochemical analyses for cultured cells were performed as described previously [8, 9] with the antibodies listed in Supporting Information Table S2. For statistical analysis of the immunocytochemical results, at least 40 colonies were examined. In the immunohistochemical analysis, the phenotypes of the grafted cells were assessed by fluorescent double immunostaining with antibodies against Venus and one of the cell-type-specific markers listed in Supporting Information Table S2. Images were obtained by fluorescence microscopy (Axioplan 2; Carl Zeiss, Thornwood, NY, www.micro-shop.zeiss.com, and BZ-9000; Keyence, Woodcliff Lake, NJ, www.keyence.com) or confocal microscopy (LSM700; Carl Zeiss).
Neurosphere Culture
Flow Cytometric Analysis
Retroviral Infection
Mouse fibroblasts transduced with Oct4-, Klf4-, Sox2-, and cMyc-expressing retroviruses were cultured in DMEM containing 10% FBS for 4 days, then trypsinized and suspended in media hormone mix (MHM) [14, 15] medium with leukemia inhibitory factor (LIF) (1,000 U/ml) and 20 ng/ml basic fibroblast growth factor (bFGF) (PeproTech Inc., Rocky Hill, NJ, www.peprotech.com/) for 14 days. In some cases, the neurospheres were dissociated and cultured at 5 104 cells per microliter in MHM with FGF2 and/or epidermal growth factor (EGF) (PeproTech). To assay the cells’ differentiation, neurospheres were plated on poly(L-ornithine)/fibronectin-coated chambers and allowed to differentiate without growth factors for 7–14 days. A pCPT-cAMP (C3912; Sigma-Aldrich, St. Louis, MO, www.sigmaaldrich.com/) was applied at 100 lM. For the comparison between two cAMP analogs, 1, 10, 100, 1,000, and 5,000 lM of pCPT-cAMP and 1, 10, 50, and 100 lM of 8-pCPT-2-O-Me-cAMP (C041; Biolog, Bremen, www.biolog.de/) were applied to culture medium. For the direct induction of NSCs from human fibroblasts, fibroblasts were purchased from Cell Applications, Inc. or obtained from skin biopsies only after both the approval of the study protocol by the Ethical committee of the Keio University (No. 20-1616) and the written informed consent of each patient (Supporting Information Table S4). The induction protocol was principally same as the mouse directly induced NSC (diNSC) induction, except that the adherent culture period was 6 days long.
Time-Lapse Observation of Neurosphere Formation To evaluate the presence of Nanog-green fluorescent protein (GFP)-positive cells throughout the neurosphere culture period, we analyzed time-lapse videos collected in a Nikon BioStation (Nikon, Tokyo, Japan, www.nikon.co.jp/) CT incubator equipped with a camera for video imaging. For these experiments, adult Nanog-GFP fibroblasts were collected 4 days after retroviral introduction of KOSM reprogramming factors and were subjected to suspension culture. During the
The Nanog-enhanced GFP (EGFP)-positive cells in neurospheres were subjected to flow cytometric analysis on a FACS Calibur. The percentage of GFP-positive cells to the total number of living cells, which were selected by the absence of propidium iodide, is presented. GFP-positive and negative cells were sorted on a FACS Vantage.
Microarray Analysis Total RNA isolation was performed with a Qiagen RNeasy Kit (http://www.qiagen.com/). DNA microarray analysis using Affymetrix Gene-Chip technology was performed as described previously [17–19]. Briefly, 100 ng of total RNA was used as a template for cDNA synthesis, and biotin-labeled cRNA was synthesized with a 30 IVT Express Kit (Affymetrix, Santa Clara, CA, www.affymetrix.com/). After generating the hybridization cocktails, hybridization to the DNA microarray (GeneChip Mouse Genome 430 2.0 Array; Affymetrix) [20] and fluorescent labeling were performed. The microarrays were then scanned with a GeneChip Scanner 3000 7G System (Affymetrix). Data analysis was carried out using Expression Console 1.1 (Affymetrix). Signal detection and quantification were performed using the MAS5 algorithm with default settings. Global normalization was performed so that the average signal intensity of all probe sets was equal to 100. For the clustering analysis, the signals were normalized and calculated by Cluster 3.0 [21], and the scores were visualized by Java Treeview [22]. The principal component analysis (PCA) was carried out by Spotfire DecisionSite 9.1.2 using normalized data.
Lentivirus Production and Infection of diNSCs A self-inactivating HIV-1-based lentivirus vector, pCSII-EFMCS-IRES2-Venus 4, was used to label NSCs for transplantation into the brain of C57/B6j mice. For lentivirus production, HEK-293T cells were transfected with pCSII-EF-MCSIRES2-Venus, pCAG-HIVgp, and pCMV-VSV-G-RSV-Rev, and the conditioned medium containing the virus particles
Matsui, Takano, Yoshida et al.
was collected. The virus was concentrated by centrifugation at 125,000g for 1.5 hours at 4 C. The concentrated viruses were added to the culture medium in which diNSCs were being formed from fibroblasts.
Transplantation The transplantation of neurospheres expressing ffLuc-cp156, a fusion protein of a fluorescent protein Venus and firefly luciferase, which had been introduced by lentiviral infection [23] was performed using a Hamilton Syringe with a stereotaxic injector, as described [24]. The needle of the Hamilton Syringe was inserted into the right striatum (2 mm lateral, 1 mm rostral to bregma; depth, 3 mm from dura) of 8-week-old female C57/b6j mice, and 3 ll of NSC suspension (2 105 cells) was injected.
Bioluminescent Imaging A Xenogen-IVIS 100 cooled CCD optical macroscopic imaging system (SC BIoScience, Tokyo, Japan, www.scbio.co.jp/
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index.html) was used for bioluminescent imaging (BLI), as reported previously [12].
Statistical Analysis The statistical significance of variations was evaluated by the unpaired two-tailed Student’s t test. All the results are presented as the mean 6 SEM.
RESULTS Generation of diNSCs from Skin Fibroblasts To obtain partially reprogrammed cells, we introduced Oct4, Sox2, c-Myc, and Klf4 (hereafter, KSOM) into adult mouse skin fibroblasts using retroviral vectors [13, 25]. These cells were then dissociated into a single-cell suspension and cultured for 14 days in the presence of LIF and FGF2 in serumfree medium, to propagate LIF-dependent primitive NSCs [26, 27], which were able to differentiate into FGF2-dependent definitive NSCs (Fig. 1A). Spheres that grew to more than 50 lm in diameter were observed 10 days after retroviral infection (Fig. 1B). To examine whether these neurospheres were clonally derived from NSCs, they were subjected to 14 days of adherent culture without growth factors. More than 60% of the spheres generated cells that expressed markers for neurons (b3-tubulin) or astrocytes (glial fibrillary acidic protein [GFAP]) (Fig. 1C). Thus, we concluded that they were neurospheres derived from NSCs, and termed them ‘‘directly induced NSCs’’ (diNSCs). Mouse ESCs can differentiate into NSCs and form neurospheres under similar floating culture conditions [26]. We previously reported that the primary neurospheres derived from mouse ESCs via embryoid bodies (EBs) mainly give rise to neurons [10]. We dissociated the same ESC line, EB3, and cultured the cells using the same protocol as for the diNSCs (from day 4 to day 18). We then differentiated these spheres on chamber slides. Neurospheres were formed similarly, but they generated few astrocytes (Fig. 1D) compared to the Figure 1. Neurosphere formation from partially reprogrammed fibroblasts. (A): Schematic of the procedure for direct neural induction. Adult mouse fibroblasts were infected with Oct4-, Sox2-, Klf4-, and c-Myc-expressing retroviruses, followed by adherent and suspension culture. (B): Process of neurosphere formation. 4F ¼ KSOM. Scale bar ¼ 100 lm. (C, D): Immunocytochemical analysis of neurospheres derived from adult mouse fibroblasts and ESCs. Scale bar ¼ 100 lm. (E--G): Immunocytochemical analysis of neural cell marker proteins in the differentiated neurospheres derived from adult mouse fibroblasts. Scale bar ¼ 50 lm. (H, I): GFP fluorescence in neurospheres generated from adult P0-Cre/LoxP-EGFP mouse fibroblasts. Phase-contrast (H) and fluorescence (I) micrographs are shown. Scale bar ¼ 100 lm. (J): Number of neurospheres obtained under different culture conditions (n ¼ 3; *, p < .001). (K, L): Immunocytochemical analysis of neural cell marker proteins in the neurospheres derived from adult mouse fibroblasts (K) and ESCs (L). Scale bar ¼ 50 lm. (M): Number of neurospheres formed within various periods of adherent culture (n ¼ 3; *, p < .05; **, p < .005; ***, p < .001). (N): Formation of secondary neurospheres. (O): Immunocytochemical analysis of neural cell marker proteins in the differentiated neural cells derived from secondary neurospheres. Scale bar ¼ 100 lm. (P): Differentiation tendency of the primary and secondary neurospheres formed by direct induction. The frequency of colonies consisting of neurons (b3-tubulin) and/or astrocytes (GFAP) was evaluated by immunocytochemistry and presented as the percentage of total colonies. Abbreviations: bFGF, basic fibroblast growth factor; EGFP, enhanced green fluorescent protein; ESCs, embryonic stem cells; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor.
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neurospheres from diNSCs. The neurospheres prepared from diNSCs could differentiate into neurons (Fig. 1E), astrocytes (Fig. 1F), and oligodendrocytes (Fig. 1G), although no oligodendrocytes were observed in the EB3-derived neurospheres. To detect any non-neural cells in differentiated diNSC-derived spheres, we performed triple-label immunostaining with antibodies against b3-tubulin, GFAP, and Nestin. We found that only 1.8% 6 1.8% of the neurosphere-derived cells did not express any of these markers (Supporting Information Fig. S1A). Therefore, this small percentage of cells was considered to be oligodendrocytes or non-neural cells. Next, we examined whether diNSCs acquired the characteristics of NSCs during reprogramming. To exclude the possibility that the diNSCs were derived from the sphere-forming multipotent neural crest stem cells that are present in the dermal skin [28–30], we made diNSC-derived neurospheres from adult P0-Cre/Floxed-EGFP mouse fibroblasts, which express EGFP in the neural crest lineage [31, 32]. The diNSC-derived neurospheres established from P0-Cre/loxP-EGFP mice showed no EGFP expression (Fig. 1H, 1I), indicating that the diNSCs did not originate from neural crest cells and were distinct from the multipotent stem cells present in dermal skin. Further detailed characterization showed that the diNSCs formed neurospheres in the presence of LIF and FGF2. The number of neurospheres was significantly reduced in the absence of LIF, and none was formed in serum-free medium that contained neither LIF nor FGF2 (Fig. 1J). When the fibroblasts used were from adult Nestin-second intronic enhancer-EGFP (Nestin-EGFP) transgenic mice [33], all the spheres derived from the diNSCs were positive for EGFP, indicating that they were neurospheres (Supporting Information Fig. S1B, S1C). To characterize the cells present in diNSC-derived spheres, we performed immunocytochemical analysis of diNSCderived neurospheres using confocal microscopy. In these neurospheres, all of the cells expressed the neural markers Nestin or b3-tubulin (Fig. 1K) to a similar extent as those derived from ESCs (Fig. 1L). These results suggested that only a few fibroblasts or non-neural cells were present in the floating diNSCderived neurospheres. The number of neurospheres peaked at 4 days in adherent culture (Fig. 1M). Dissociated diNSC-derived primary neurospheres generated secondary neurospheres (Fig. 1N) that gave rise to neurons and astrocytes (Fig. 1O). These secondary neurospheres were highly astrogenic (Fig. 1P) and resembled the mature NSCs that appear in late embryonic stages in vivo [34]. These data suggest that diNSCs can self-renew, a characteristic of NSCs, and gradually develop into mature multipotent NSCs in vitro.
Cell Type of Origin and Reprogramming Duration Influence the Differentiation Properties of the diNSCs We next tested whether the somatic cell of origin or reprogramming procedure influenced the properties of the diNSCs. First, we compared the differentiation properties of neurospheres generated from adult cells grown for various periods in adherent cultures after reprogramming factors had been introduced (Fig. 1M). Although longer periods of adherent culture increased the number of neurospheres (Fig. 1M), these neurospheres were neurogenic and not gliogenic (Fig. 2A). This result suggested that the duration of reprogramming affected the maturity of the diNSCs. We then compared the differentiation properties of diNSCs derived from mouse embryonic fibroblasts (MEFs) and adult mouse fibroblasts. Neurospheres derived from MEFs generated mainly neurons and few astrocytes, as did neurospheres derived from dissociated EB3 cells and 38C2-iPSC (Fig. 2B) [35], whereas those
Direct Induction of Rapidly Differentiating NSCs
Figure 2. Properties of directly induced NSCs. (A, B): Effect of the adherent-culture duration (A) and cell source (B) on the differentiation tendency of directly induced neurospheres. Differentiated neurospheres were subjected to immunocytochemical analysis for b3-tubulin (neurons) and GFAP (astrocytes). The frequency of colonies consisting of neurons and/or astrocytes is presented as the percentage of total colonies. (C--F): GFP fluorescence in neurospheres generated from NanogEGFP adult mouse fibroblasts (C, D) or MEFs (E, F). Phase-contrast (C, E) and fluorescence (D, F) micrographs are shown. Scale bar ¼ 200 lm. (G): Flow cytometric analysis of the Nanog-EGFP-positive cells in neurospheres established from Nanog-EGFP adult mouse fibroblasts or MEFs (n ¼ 3; *, p < .001). (H): Representative fluorescenceactivated cell sorting plots for cells in the neurospheres from NanogEGFP MEFs or adult fibroblasts. (I, J): Methylation status of the CpGs in the Stat3 recognition sequence of the GFAP promoter (I) (n ¼ 10; *, p < .005) or in the Oct4 promoter region (J) (n ¼ 10; *, p < .001). The proportion of methylated cytosines is shown. Abbreviations: EGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; iPSC, induced pluripotent stem cell; MEFs, mouse embryonic fibroblasts.
generated from adult fibroblasts generated relatively more astrocytes. Next, to monitor the residual pluripotent cells, we generated diNSC-derived neurospheres from adult and embryonic fibroblasts established from Nanog-GFP transgenic mice (a gift from Dr. Shinya Yamanaka’s laboratory) [35]. Although both types of fibroblasts generated neurospheres, only 12.5% of the neurospheres from adult fibroblasts showed visible Nanog-GFP fluorescence, but 93.9% of the ones from MEFs did (Fig. 2C-2F). Quantitative analysis by fluorescence-activated cell sorting (FACS) showed 10 times more Nanog-GFP-positive
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cells in the MEF-derived neurospheres than in the adultderived ones (Fig. 2G, 2H). To determine whether the NSCs were generated from Nanog-GFP-positive or -negative cells, we first examined whether Nanog-GFP-positive cells were present on day 4 by using flow cytometric analysis. Consistent with a previous report [35], no GFPþ cells were found on day 4 (Supporting Information Fig. S2), indicating that sphereforming cells were not derived from fully reprogrammed iPSCs at the start of the neurosphere culture. However, it is possible that a small number of cells were gradually reprogrammed into fully pluripotent GFPþ cells and then committed to neural lineages that led to neurosphere formation during the floating culture period. Alternatively, a small number of cells in the neurospheres may have retained or acquired the pluripotency in this neurosphere culture system. To investigate whether or not the neurospheres were generated from Nanog-GFP-positive cells, we performed a continuous observation using time-lapse microscopy (Supporting Information Movie S1). We observed more than 100 spheres and found none of the observed neurospheres expressed GFP fluorescence by day 10 of sphere formation. However, 13 of 122 neurospheres contained a small number of visible Nanog-GFP-positive cells by day 14. Therefore, we concluded that diNSCs were generated mainly from nonpluripotent cells in this culture system. However, it is possible that a small number of diNSCs were also generated from pluripotent cells or that a small number of cells within the neurospheres were reprogrammed to be pluripotent cells during neurosphere formation. We next examined the methylation within a STAT3-binding site in the GFAP promoter region using bisulfite PCR and sequencing, because this site is demethylated gradually in NSCs during development [36, 37]. The diNSC-derived neurospheres established from adult mouse fibroblasts had significantly less methylation (49.2%) in the GFAP promoter region than did adult mouse fibroblasts (78.3%) or neurospheres derived from dissociated 38C2-iPSCs in the presence of LIF and FGF (82.0%) (Fig. 2I). The amount of methylation was also lower than in tertiary neurospheres derived from ESCs via EBs [10, 37]. We also examined the methylation in the promoter region of the pluripotent marker gene, Oct4, because it is demethylated in iPSCs [13, 35]. The Oct4 promoter region was highly methylated in the neurospheres derived from adult fibroblasts compared to the LIF- and FGF2-dependent primary neurospheres from dissociated 38C2-iPSCs (Fig. 2J). These results together suggested that the differentiation properties of diNSCs are determined by the cell source and the culture period after the introduction of pluripotent genes, which could affect the state of reprogramming.
Optimization of Culture Conditions to Increase the Efficiency of diNSC Induction Although we succeeded in making neurospheres derived from diNSCs, less than 0.01% of the dissociated fibroblasts treated with Yamanaka factors generated neurospheres. To increase the efficiency of neurosphere formation, we used cAMP analog, pCPT-cAMP (Sigma; C3912), which increases the survival of LIF-dependent primitive NSCs by inhibiting their apoptosis [27]. We added pCPT-cAMP at various concentrations to the serum-free medium used for the suspension cultivation step and found that 100 lM of pCPT-cAMP most efficiently induced neurosphere formation (to 0.03%) (Fig. 3A--3D; Supporting Information Fig. S3A). Although the diameters of the neurospheres were similar to those that formed without cAMP (Fig. 3E), the frequency of gliogenic neurospheres was significantly lower than in cultures without pCPT-cAMP (Fig. 3F). Thus, we removed pCPT-cAMP from the medium on day 6, www.StemCells.com
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Figure 3. Transient pCPT-cAMP treatment facilitates mature neurosphere formation. (A): Schematic of culture method using pCPTcAMP. pCPT-cAMP was added to the serum-free medium and removed after 2 days in groups C and D. In groups C and D, the neurospheres were subjected to one or two dissociations. 4F ¼ KSOM. (B, C): Neurosphere formation after 14 days of suspension culture without (B) or with (C) pCPT-cAMP (scale bar ¼ 100 lm). (D, E): Average sphere formation percentage and diameter of neurospheres in cultures without pCPT-cAMP (n ¼ 17), with pCPT-cAMP (n ¼ 7), with pCPT-cAMP and one passage (n ¼ 3), and with pCPT-cAMP and two passages (n ¼ 3) (*, p < .05). (F): Differentiation potential of neurospheres under various conditions. Differentiated neurospheres were subjected to immunocytochemical analysis for b3-tubulin (neurons) and glial fibrillary acidic protein (astrocytes). The frequency of colonies consisting of neurons and/or astrocytes is presented as the percentage of total colonies. Abbreviations: bFGF, basic fibroblast growth factor; FBS, fetal bovine serum; LIF, leukemia inhibitory factor.
and dissociated the neurospheres to enhance the differentiation of NSCs (Fig. 3A). The diNSC-derived neurosphere formation ratio increased to 2.98% in this condition (Fig. 3D). The addition of pCPT-cAMP for 48 hours and additional passages resulted in increased numbers and frequencies of astrogenic neurospheres derived from the diNSCs (Fig. 3F). These frequencies were similar to those of LIF-dependent neurospheres derived from dissociated ESCs and subjected to directed differentiation [26, 27], even though our starting cells were fibroblasts. Different cAMP analogs are known to activate either the Epac or protein kinase A (PKA) pathway [38, 39]. To determine which pathway was crucial for the increase in neurosphere formation efficiency, we tested the effects of 8-pCPT-2-O-Me-cAMP (Biolog C041), a distinct cAMP analog that activates only the Epac pathway. While pCPT-cAMP increased neurosphere formation efficiency at 100 mM (p < .05), 8-pCPT-2-O-Me-cAMP did not increase the number of spheres at any concentration tested (Supporting Information Fig. S3B). This result suggests that the PKA pathway is involved in the survival of diNSCs.
diNSCs Respond to EGF Treatment in Early Passages To use iPS-derived NSCs for transplantation, it is important to choose safe iPS clones that do not form teratomas in host animals [8, 9]. We previously reported that the safety of iPSCs can be monitored by the expression of pluripotent markers, and that neurospheres containing less than 0.018% Nanog-GFPpositive cells do not form teratomas after their transplantation [8]. To decrease the Nanog-GFP-positive cells in the diNSCderived neurospheres, we tried to withdraw LIF from the culture medium during the induction of diNSC-derived neurospheres from the fibroblasts of Nanog-GFP mice. Although the frequency of green neurospheres decreased dramatically with
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tially reprogrammed cells and not from fully reprogrammed, pluripotent cells.
Microarray Analysis of diNSC-Derived Neurospheres
Figure 4. Adult fibroblast-derived directly induced neural stem cells respond to EGF. (A): Schematic of EGF introduction and LIF withdrawal in the suspension culture period. 4F ¼ KSOM. (B, C): Neurospheres established in the presence of LIF for 14 days. (D, E): Neurospheres established in the presence of LIF for 10 days and cultured 4 days with EGF and without LIF. Phase-contrast (B, D) and fluorescence (C, E) micrographs are shown. (F): The percentage of Nanog-EGFPþ cells in directly induced neurospheres under various culture conditions. The red bar indicates 0.018%. (G): Flow cytometric analysis of neurospheres established from Nanog-EGFP mouse adult fibroblasts. Nanog-EGFPþ cells were almost completely lost after LIF withdrawal and EGF treatment. (H): Neurosphere formation under the EGF condition. Five kinds of cells were subjected to direct neural induction with EGF treatment and LIF withdrawal. Abbreviations: APC, adenomatous polyposis coli; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; EGFP, enhanced green fluorescent protein; ESCs, embryonic stem cells; FBS, fetal bovine serum; iPSCs, induced pluripotent stem cells; MEF, mouse embryonic fibroblast; LIF, leukemia inhibitory factor.
LIF withdrawal, the frequency of neurosphere formation in this condition was as low as 0.02%. To solve this problem, we added EGF to the neurosphere culture, because mature NSCs respond to EGF by forming neurospheres [26, 40]. We used EGF instead of LIF on days 11–15 in the suspension culture period (Fig. 4A). In this condition, the frequency of diNSCderived neurosphere formation increased to 1%, with reduced visible green fluorescence (Fig. 4B--4E). Using culture conditions 1-4 presented in Figure 4A, more than 0.15% of the undifferentiated cells in the neurospheres was Nanog-GFP positive, by flow cytometric analysis (Fig. 4F). However, we observed a great reduction in the percentage of Nanog-EGFP-positive cells in neurospheres cultured with EGF (0.018%) had teratomas (Supporting Information Fig. S8). Finally, EGF-dependent diNSCs successfully differentiated into neurons, astrocytes, and oligodendrocytes in host animals (Fig. 6D).
Establishment of diNSCs from Human Adult Fibroblasts
Figure 6. In vivo engraftment and differentiation of directly induced neurospheres. (A): IVIS images demonstrating the engraftment of directly induced neurospheres in the striatum. Directly induced neurospheres transduced with luciferase were injected into the striatum and observed at postoperation day 28. (B): Bright-field image of directly induced neural stem cell (diNSC)-derived neurospheres engrafted into the brain of a transplanted mouse, stained with antibodies to GFP, detected with 3,30 -diaminobenzidine (brown). Scale bar ¼ 1 mm. (C): Immunohistochemical analysis showing the engraftment of diNSC-derived neurospheres. Venus-positive transplanted cells were detected by anti-GFP antibody. Scale bar ¼ 50 mm. (D): Immunohistochemical analysis of markers for neurons (Hu), astrocytes (GFAP), and oligodendrocytes (APC) in the C57b6j mouse striatum, 4 weeks after the transplantation of directly induced neurospheres. Scale bar ¼ 10 lm. Abbreviations: APC, adenomatous polyposis coli; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein.
mouse fibroblasts were infected with pMX retroviral vectors carrying five genes, KSOM and GFP. Four days later, adherent fibroblasts infected with the five genes (KSOM þ GFP) were selected by their fluorescence using a flow cytometer, dissociated, and cultivated in floating culture. At this time, 61.2% of the fibroblasts was GFP-positive. Clonal neurospheres were generated only from the GFP-positive fraction. However, 14.4% of the total cells in these neurospheres was GFP-positive (Supporting Information Fig. S6A, S6B). Quantitative PCR analysis also confirmed that all of the retrovirusinduced transgenes were decreased, but not completely silenced, in diNSC-derived spheres at day 14 of suspension (day 18) (Supporting Information Fig. S6C) [13].
Finally, we applied our method to the establishment of diNSCs from adult human fibroblasts (Fig. 7A). We introduced human Oct4, Sox2, c-Myc, and Klf4 (KSOM) into adult human fibroblasts (Fig. 7B) using retroviral vectors. These cells were then dissociated into single-cell suspensions and cultured for 14 days in the presence of LIF and FGF2 in serum-free medium. pCPTcAMP treatment was performed for 10 days. After 14 days of suspension culture, a few spheres were observed (Fig. 7C) and subjected to 14 days of adherent culture without adding exogenous growth factors (Fig. 7D, 7E). Four human fibroblast lines were tested in this experiment. The fibroblasts derived from a 16-year-old female demonstrated the highest sphere formation efficiency (Fig. 7F; Supporting Information Table S4). Formed spheres were allowed to differentiate using adherent culture conditions. These cells expressed b3-tubulin (Fig. 7D) or GFAP (Fig. 7E), indicating that the presence of neurons and astrocytes. The frequency of b3-tubulin-positive cells was 58.0% 6 12.4%, and that of GFAP-positive cells was 8.0% 6 3.74% (Fig. 7G). These data suggested that the majority of cells were neural cells. We also analyzed Oct4 expression to detect pluripotent cells. 2.58% 6 2.56% (Fig. 7G) of the cells was Oct4 positive (Supporting Information Fig. S9).
DISCUSSION One of the major advantages of iPSC technology is that it allows for the creation of cells that are genetically matched to patients. However, in SCI animal models, the transplantation of NSCs is not effective at the chronic stage, after glial scar formation [53], which occurs in humans within a few weeks after the injury. Rather, it is believed that a curative effect
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Figure 7. Directly induced neural stem cells induced from adult human fibroblasts. (A): Schematic of the method for direct neural induction of human fibroblasts. Adult human fibroblasts were retrovirally transduced with Oct4, Sox2, Klf4, and c-Myc, followed by adherent and suspension culture, with growth factors. 4F ¼ KSOM. (B): Human fibroblasts before the introduction of four reprogramming factors. Scale bar ¼ 100 lm. (C): Neurospheres derived from adult human fibroblasts. Scale bar ¼ 100 lm. (D, E): Immunocytochemical analysis for b3-tubulin and GFAP in the differentiated neurospheres derived from adult human fibroblasts. Scale bar ¼ 50 lm. (F): The efficiency of neurosphere formation from various lines of fibroblasts (n ¼ 3). (G): Immunocytochemical analysis of neural cell or pluripotent cell marker proteins in the differentiated neurospheres derived from adult human fibroblasts. The percentage of cells positive for each indicated marker is shown. Abbreviations: bFGF, basic fibroblast growth factor; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor.
may be achieved if the NSC transplantation is done within the subacute phase (approximately 7–14 days after injury) [12], due to the changes in the microenvironment within the injured spinal cord [54, 55], and that glial cells derived from these NSCs can play important roles in restoring neural functions to the host animals [9, 11, 54]. Furthermore, the safety of each iPS clone should be thoroughly evaluated before it is used for cell therapy, because of variations in their propensity to form teratomas [8]. Therefore, it is presently impossible to propagate safe NSCs derived from iPSCs made from the injured patient’s own cells, within the subacute phase of SCI. We found that the diNSCs derived from adult fibroblasts generated astrocytes similar to the NSCs present within the striatum of the midgestation mouse embryo [36, 37, 55, 56]. Gliogenic NSCs are currently considered the best source for cell therapy for SCI [11]. Using our method, fibroblasts were differentiated into diNSC-derived neural cells and amplified more than 133 times within 18 days after their infection with retrovirus. In addition, even though they were generated from adult fibroblasts, the diNSCs contained as few Nanog-GFP-positive pluripotent cells as the NSCs from MEF-derived iPS clones. When we generated secondary neurospheres from adult tail tip fibroblast-derived iPSCs via EBs, all of the clones contained Nanog-GFP-positive pluripotent cells detectable by FACS analysis. However, the numbers of residual NanogGFP-positive cells in diNSC-derived neurospheres grown in EGF alone were less (
