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Figures
- Fig. 1.
Direct reprogramming of fibroblasts into LD-iNSCs. (A) Schematic representation of the doxycycline (Dox)-inducible lentiviral polycistronic vectors. The vector consists of a polycistronic gene that contains the human cDNA sequences of OCT4, KLF4, SOX2, L-MYC and NANOG, bound by self-cleaving 2A elements (2A) and IRES, and driven by the TRE3G promoter, which is activated by the binding of the Tet-On 3G transactivator in the presence of Dox in a culture medium. (B) Schematic of the experimental setup and strategy for the generation of LIF-dependent induced neural progenitors (LD-iNSCs). (C) Colony morphology of LD-iNSCs exhibited a tightly packed structure similar to primary neural progenitors. (D) RT-PCR showed that LD-iNSCs expressed the neural progenitor markers NESTIN, SOX1 and PAX6. GAPDH was used as an internal control. RNA was extracted from three LD-iNSC clones (LD-iNSC#5, LD-iNSC#18 and LD-iNSC#42.2), uninfected parent fibroblasts (negative control), and from human iPSCs. (E) LD-iNSCs cultured on feeder cells in the presence of 2i/LIF tested positive for SOX1 (red) and PAX6 (green). (F) The population doubling level (PDL) of LD-iNSCs in comparison with human iPSCs. (G) A representative karyotype of LD-iNSCs at passage 28. (H) Ectopic factor dependence of LD-iNSCs. Upon doxycycline withdrawal, LD-iNSC colony morphology was lost, and the cells assumed a square morphology. The subsequent addition of Dox reversibly produced LD-iNSCs. Days of differentiation or reprogramming are indicated. Scale bars are 500 μm (C and H).
- Fig. 2.
Characterization of LD-iNSCs. (A) Immunostaining for OCT4, SOX2 and NANOG in LD-iNSCs with (top) or without (bottom) Dox treatment. Only SOX2 expression was detected in LD-iNSCs without Dox (bottom). Cell nuclei were visualized with DAPI. (B) RT-PCR showed that LD-iNSCs expressed markers of pluripotency, such as endogenous OCT4, NANOG, SOX2, LIN28, REX1, LEFTY1 and TERT. GAPDH served as the internal control. RNA was extracted from three LD-iNSC clones (LD-iNSC#5, LD-iNSC#18 and LD-iNSC#42.2), uninfected parent fibroblasts (negative control), and from human iPSCs (positive control). (C) DNA methylation status of CpG islands in the OCT4, REX1 and NANOG promoter regions was assessed by bisulfite sequencing PCR. Open circles indicate unmethylated, and filled circles indicate methylated, CpG dinucleotides. Representative sequences from three LD-iNSC clones (LD-iNSC#5, LD-iNSC#18 and LD-iNSC#42.2), uninfected parent fibroblasts (negative control) and human iPSCs (positive control) are shown. The percentage of CpG methylation in each CpG island within the respective cell line is indicated. (D) Expression levels of pluripotent marker genes (OCT4, REX1 and NANOG) selected in (C). All data are normalized to hiPSCs (positive control), whose expression was assumed to be 1.0 for genes in other cell lines. (E) ChIP-qPCR analysis of the presence of histone 3 lysine 4 marker in the promoter region of the pluripotency gene OCT4 in LD-iNSC#18, uninfected parent fibroblasts (negative control), and human iPSCs (positive control). Error bars are mean±s.e.m., n=3.
- Fig. 3.
Expression of neural progenitor markers in LD-iNSCs. (A) A phase-contrast image of LD-iNSCs cultured onto poly-L-lysine and laminin-coated dishes under feeder-free conditions in the presence of 2i/LIF. A high-magnification image of cells is also shown in the inset image. (B-E) LD-iNSCs without feeder cells in the presence of 2i/LIF tested positive for NESTIN (D, green), FABP7 (E, red), SOX1 (F, green) and SOX2 (G, red). DAPI staining is shown in blue. (F) Immunostaining for Ki67 (red) in LD-iNSCs without feeder cells in the presence of 2i/LIF. DAPI staining is shown in blue. (G-I) An immunofluorescence assay for the detection of markers of neurons (TUJ1), astrocytes (GFAP) and oligodendrocytes (O4). LD-iNSCs have the capacity to differentiate into neurons, astrocytes and oligodendrocytes in vitro. (J) Representative image of an oligodendroglial morphology. Scale bars are 200 μm (A-F) or 100 μm (G-I).
- Fig. 4.
Differentiation of LD-iNSC into motor neurons. (A) Schematic representation of the differentiation of LD-iNSCs into motor neurons. (B) LD-iNSC-derived motor neurons were positive for TUJ1 and expressed motor neuron markers such as HB9, ISLET1, HOXC8 and CHAT. Cell nuclei were visualized with DAPI. Scale bars are 100 μm. (C) RT-PCR revealed increased/induced expression of postmitotic markers of motor neurons (CHAT, HOXA5 and HOXC5) in LD-iNSCs-derived motor neurons in contrast to LD-iNSCs and human fibroblasts. GAPDH served as the internal control. Abbreviations: BDNF, brain-derived neurotrophic factor; GDNF, glial cell-derived neurotrophic factor.
- Fig. 5.
Differentiation of LD-iNSCs into dopaminergic neurons. (A) An overview of the protocol for differentiation of LD-iNSCs into dopaminergic neurons. (B-F) Efficient production of dopaminergic neurons from LD-iNSCs was demonstrated by immunostaining for EN-1, FOXA2, LMX1A, NURR1 and TH (red). TUJ1 is a pan-neuronal marker used to assess the total production of neurons (green). Cell nuclei were visualized with DAPI. Scale bars are 100 μm (B-F). Abbreviations: FGF8b, fibroblast growth factor 8 b; IGF-1, insulin-like growth factor-l; TGF-β3, transforming growth factor beta 3; db-cAMP, dibutyryl cyclic adenosine monophosphate. (G) Percentage of TH+ neurons among total neurons (TUJ1+) differentiated from LD-iNSCs and hESC-derived NSCs under DA neuronal differentiation conditions was determined by immunofluorescence. Error bars are mean±s.e.m., n=6.
- Fig. 6.
Conversion of LD-iNSCs to hiPSCs. (A) Schematic representation of the conversion of LD-iNSCs to a stable pluripotent state. (B) Three days after switching to the standard hESC medium supplemented with bFGF, LD-iNSC lost its colony morphology (top panel) and the cells adopted a fibroblast-like appearance (middle panel). After further growth in this culture condition, LD-iNSCs were converted to hiPSCs (bottom panel). The converted cells (LD5F-hiPSCs) exhibited hESC-like colony morphology. (C) Typical ESC markers (OCT4, NANOG and SOX2) were expressed in LD5-hiPSCs, according to immunofluorescence analysis. Other pluripotent cell surface markers (SSEA4 and TRA-1-81) were also detected by immunofluorescence staining. DAPI was used to visualize the cell nuclei. (D) Injection of undifferentiated LD5F-hiPSCs into immune-deficient mice led to the formation of teratomas containing derivatives of all three germ layers: cartilage (mesoderm, left panel), pigmented retinal epithelium (ectoderm, middle panel) and gut-like epithelium (endoderm, right panel).
- Fig. 7.
Introduction of a transgene into LD-iNSCs. (A) Results of the electroporation experiments with LD-NPs and hiPSCs are summarized in the table (left panel). The right panel shows representative images of LD-iNSCs after electroporation of an EGFP expression vector (15 kb). As shown in the table, the percentage of EGFP-positive LD-iNSC colonies was approximately 50% under our transfection conditions, but negligible or 0% in conventional hiPSCs. (B) Conversion of EGFP-positive LD-iNSCs to a stable pluripotent state. hiPSCs converted from an EGFP-positive LD-iNSC clone were obtained at the indicated time point. mESCs/iPSCs have generally demonstrated a relatively high level of transfection efficiency, whereas hESCs/iPSCs are notoriously difficult to transfect (Cao et al., 2010). Therefore, these results suggest that the conversion to hiPSCs might be a novel method that could increase the transfection efficiency of hiPSCs.
- Fig. 8.
LIF-dependent primitive NSCs are obtained in the process of reprogramming to iPSCs. (A) The establishment of various desired neuronal types by means of multipotent LD-iNSCs. LD-iNSCs are directly derived from human fibroblasts via continuous expression of reprogramming factors in the presence of 2i/LIF in the culture medium. LD-iNSCs retain low levels of OCT4 and NANOG expression, but high levels of SOX2 expression, have self-renewal capacity, and can differentiate into various neuronal types (such as motor neurons and dopaminergic neurons), astrocytes and oligodendrocytes. In addition, pluripotent stem cells can be derived by simple re-plating of LD-iNSCs in hESC medium supplemented with bFGF, suggesting that LD-iNSCs are derived from intermediate partially reprogrammed cells. (B) Pluripotency levels induced by in vitro reprogramming correlate significantly with the extrinsic signaling environment. The delivery of reprogramming factors into primary human fibroblasts, and culturing of the cells in the presence of 2i/LIF leads to the resulting cell lines (such as LD-iNSCs) resembling multipotent primitive neural stem/progenitor cells (Hirano et al., 2012). Aside from the 2i/LIF treatment, bFGF signaling plays an important role in the derivation and maintenance of hiPSCs (a primed state). Fully naïve hiPSCs were recently derived using a novel medium called naïve human stem cell medium (NHSM) (Gafni et al., 2013). Taken together, the combination of reprogramming factors and culture supplements determines the respective pluripotent states.
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