Mononuclear cells (MNCs) were harvested from hUCB using Lymphoprep (Stem Cell Technologies) density-gradient centrifugation. The isolation and research protocols were approved by the Boramae Hospital Institutional Review Board (IRB) and the IRB of Seoul National University (1109/001-006). Isolated mononuclear cells were subject to magnetic bead selection for purification of CD34⁺ cells using a CD34 microbeads kit (Miltenyl Biotec) following the manufacturer’s instructions. The purity of the isolated cells was assessed through flow cytometry. hUCB CD34⁺ cells were maintained in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum, 50 ng/ml SCF, 100 ng/ml Flt3L, 50 ng/ml TPO and 20 ng/ml IL-6 before infection. All cytokines were purchased from R&D Systems.

The cells were washed with PBS, incubated with anti-CD34-FITC antibody (BD Biosciences) for 30 min at 4°C. After washing, cells were resuspended in 500 μl of PBS and analyzed using FACSCalibur (BD Biosciences).

Viral production and infection were performed as previously described (13). The retroviral pMX-SOX2 and pMX-HMGA2 vectors were packaged in HEK293 FT cells (Invitrogen) using FuGENE 6 (Roche). hUCB CD34⁺ cells were infected with retroviruses for 2 rounds in the hematopoietic cells growth medium. With day 0 designated as the time of first infection, the medium was changed to ReNcell NSC maintenance media (Millipore) containing 20 ng/ml bFGF and 20 ng/ml EGF on day 3. All cytokines were purchased from R&D Systems. As positive control for human neural stem cells, H9 hESC-derived NSCs (H9-NSCs, Invitrogen) were cultured using the ReNcell media.

Cells were fixed in 4% paraformaldehyde (PFA, in PBS) for 20 min at room temperature, permeabilized with 0.25% Triton X-100 for 10 minutes, incubated with 5% normal goat serum for 1 hour and incubated with primary antibodies overnight at 4°C. Subsequently, the cells were incubated with secondary Alexa 488- or 594-labeled antibodies (Invitrogen) for 1 hour at room temperature. The nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 minutes. The images were taken using a confocal microscope (Eclipse TE200, Nikon). The details about primary antibodies are listed in Table 1.

Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions. cDNA was synthesized by reverse transcription using the Superscript III First-Strand Synthesis System (Invitrogen). qPCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems). The following primers were used: SOX2 (F: AGTCTCCAAGCGACGAAAAA, R: TTTCACGTTTGCAACTGTCC), PAX6 (F: CAGCTCGGTGGTGTCTTTG, R: AGTCGCTACTCTCGGTTTA), NESTIN (F: AACAGCGACGGAGGTCTCTA, R: TTCTCTTGTCCCGCAGACTT), GLAST (F: CTCACAGTCACCGCTGTCAT, R: ACTATCTAGCGCCGCCATTC), OLIG2 (F: CCGATGACCTTTTTCTGCCG, R: CCACTGCCTCCTAGCTTGTC), OCT4 (F: GACAGGGGGAGGGGAGGAGCTAGG, R: CTTCCCTCCAACCAGTTGCCCCAAAC), NANOG (F: AATAACCTTGGCTGCCGTCTC, R: AGCCTCCCAATCCCAAACAAT).

For differentiation and monolayer culture, hUCB iNSCs were seeded onto poly-L-ornithine/fibronectin (PLO/FN)-coated plates. Neural differentiation medium consisted of DMEM/F12 and Neurobasal medium (1:1), 2 mM GlutaMAX, 1% Penicillin-streptomycin, 1 μM Retinoic acid, 20 ng/ml BDNF, 20 ng/ml GDNF, 200 μM Ascorbic acid, and 5 μM Forskolin. For oligodendrocyte differentiation, cells were cultured in induction medium #1 which consists of DMEM/F12, 1% N2, 100× MEM-NEAA, 1 μM Retinoic acid, 2 μM purmorphamine, 2 mM GlutaMAX and 10 ng/ml bFGF for 1 week. Subsequently, induction medium #2 containing DMEM/F12, 0.5× N2, 2% B27, 100× MEM-NEAA, 30 ng/ml T3, 1 mM cAMP, 2 μM purmorphamine, 20 ng/ml PDGF, 100 ng/ml IGF, and 10 ng/ml NT3 was used for 2 weeks. For astrocyte differentiation, EGF and bFGF were removed from NSC maintenance medium and 1% FBS was added.

In order to generate iNSCs from hUCB, we first attempted to isolate MNCs from hUCB (Fig. 1A). As HetaSep sediments red blood cells (RBCs) by aggregation, HetaSep solution was mixed with hUCB samples at the ratio of 1:5. Subsequently, we applied Lymphoprep density gradient method to culture MNCs. After centrifugation, MNCs in between upper plasma and bottom Lymphoprep layers were harvested (Fig. 1B). We used typical magnetic bead for positive selection of CD34⁺ cells from MNCs and purified cells were maintained in hematopoietic stem cells growth medium for 3 days (Fig. 1C). At day 1 of derivation, the purity was approximately 85~90% (Fig. 1D). The conversion process was induced by two rounds of transduction of retroviral SOX2 and HMGA2 into hUCB CD34⁺ cells. We supplemented cytokines (SCF, Flt3L, TPO, and IL-6) in virus-containing medium to activate the proliferation of the population of hematopoietic stem cells, as retrovirus are transduced only into dividing cells. After the transduction, cells were cultured in IMDM with the cytokines for 24 h to further stimulate the proliferation. To re-plate suspended hUCB CD34⁺ cells on feeder cells, we inactivated STO cells with mitomycin C and seeded them on gelatin-coated plates following a conventional feeder-dependent iPSC generation protocol (Fig. 1E). After 1 day, the medium was replaced with hNSC medium and replenished every day until iNSC-like colonies appear. We have found that although colonies appear as early as in 7 days, it is better to sustain the proliferation of colonies for more than 14 days for the ease of the colony-picking step (Fig. 1F and 1G). Finally, iNSC colonies were picked in small pieces using a pasture pipette and transferred to non-coated plates for homogeneous culture of neurospheres.

In order to determine whether the hUCB iNSCs have clonal expansion properties like human neural stem cells, we attempted to sub-culture neurospheres derived from hUCB iNSC colonies. The neurospheres derived from initial iNSC-like colonies were transferred on PLO/FN-coated plates for monolayer culture (Fig. 1H). When the cells reached 80~90% confluence, they were detached using Accutase, and the neurosphere culture step was repeated at least three times for the establishment of homogeneous hUCB iNSC lines (Fig. 1I).

After passage 6, we briefly performed immunostaining with neural stem cell markers, PAX6 and NESTIN, for the selection of fully converted hUCB iNSCs. We have found that hUCB iNSC lines have different characteristics depending on their original colonies and that some cell lines show weak expression of neural stem cell markers. Upon passaging, immunostaining and qRT-PCR for diverse neural stem cell markers were performed, and activation of endogenous genes was confirmed before proceeding into further experiments. As shown in Fig. 2A, hUCB iNSCs prominently expressed neural stem cell-specific markers, including SOX2, NESTIN, and PAX6. Furthermore, hUCB iNSCs were immunoreactive for proliferation marker Ki67. qRT-PCR analysis showed that both two cell lines (hUCB iNSC #7 and hUCB iNSC #8) expressed endogenous NSC markers; SOX2, NESTIN, GLAST, BLBP, PAX6, and OLIG2 (Fig. 2B). The expression levels of NSC markers were indistinguishable from H9-NSCs, suggesting that hUCB iNSCs were properly reprogrammed. Subsequently, we investigated whether the established hUCB iNSCs express pluripotent cell markers. qRT-PCR results showed that OCT4 and NANOG were not expressed in hUCB-iNSCs, suggesting that pluripotent cells are not contained in hUCB-iNSCs (Fig. 2C).

To verify that the hUCB iNSCs were capable of differentiating into neurons, astrocytes and oligodendrocytes in vitro, we plated the cells onto PLO/FN-coated coverslips in 24 well culture plate. Withdrawal of bFGF and EGF and change into tri-lineage differentiation medium induced the differentiation of iNSCs. Accordingly, the culturing of hUCB iNSCs in neuronal differentiation media supported the generation of neurons, which expressed neuron-specific marker Neurofilament (NF) (Fig. 3A). Moreover, exposure of hUCB iNSCs to astrocyte differentiation media resulted in the formation of astrocytes that expressed GFAP (Fig. 3B). In addition, hUCB iNSCs could differentiate into O4- and OLIG2-positive oligodendrocytes (Fig. 3C). Thus, hUCB iNSCs are tripotent and can generate neurons, astrocytes, and oligodendrocytes in vitro.