Int J Stem Cells.  2017 Nov;10(2):227-234. 10.15283/ijsc17025.

Direct Conversion of Human Umbilical Cord Blood into Induced Neural Stem Cells with SOX2 and HMGA2

• Kim JJ
• Shin JH
• Yu KR
• Lee BC
• Kang I
• Lee JY
• Kim DH
• Seo Y
• Kim HS
• Choi SW
• Kang KS

• KMID: 2400876
• DOI: http://doi.org/10.15283/ijsc17025

Abstract

Recent advances have shown the direct reprogramming of mouse and human fibroblasts into induced neural stem cells (iNSCs) without passing through an intermediate pluripotent state. Thus, direct reprogramming strategy possibly provides a safe and homogeneous cellular platform. However, the applications of iNSCs for regenerative medicine are limited by the restricted availability of cell sources. Human umbilical cord blood (hUCB) cells hold great potential in that immunotyped hUCB units can be immediately obtained from public banks. Moreover, hUCB samples do not require invasive procedures during collection or an extensive culture period prior to reprogramming. We recently reported that somatic cells can be directly converted into iNSCs with high efficiency and a short turnaround time. Here, we describe the detailed method for the generation of iNSCs derived from hUCB (hUCB iNSCs) using the lineage-specific transcription factors SOX2 and HMGA2. The protocol for deriving iNSC-like colonies takes 1~2 weeks and establishment of homogenous hUCB iNSCs takes additional 2 weeks. Established hUCB iNSCs are clonally expandable and multipotent producing neurons and glia. Our study provides an accessible method for generating hUCB iNSCs, contributing development of in vitro neuropathological model systems.

Keyword

Human umbilical cord blood; Induced neural stem cells; Reprogramming; Direct conversion

MeSH Terms

Animals
Fetal Blood*
Fibroblasts
Humans*
In Vitro Techniques
Methods
Mice
Neural Stem Cells*
Neuroglia
Neurons
Regenerative Medicine
Transcription Factors
Umbilical Cord*
Transcription Factors

Figure

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  Fig. 1 Derivation of hUCB iNSCs. (A) Schematic presentation of protocols for hUCB iNSCs generation. hUCB CD34+ cells are isolated and expanded with proliferation-stimulating cytokines for 2~3 d and transduced with SOX2 and HMGA2 retrovirus. Distinct iNSC colonies appear after 7~10 d and are picked for repetitive sphere culture and monolayer culture for 10~14 d. (B) Before centrifugation, the upper layer is RBCs-reduced hUCB and the bottom layer is Lymphoprep (left). After centrifugation, a thin layer containing mononuclear cells appears between upper plasma layer and bottom Lymphoprep layer (right). (C) Phase contrast images of hUCB CD34+ cells cultured with cytokines at day 0 (left) and day 3 (right). Scale bars, 200 μm. (D) Flow cytometric analysis of hUCB CD34+ cells cultured for 1 d with cytokines. The cells were 90.88% positive for CD34. (E) Representative image of hUCB CD34+ cells seeded on STO feeder cells. (F, G) Example of hUCB iNSC colonies cultured in neural stem cell inductive medium for 7 d (F) and 12 d (G). (H) Morphology of neurospheres derived from initial hUCB iNSC colonies (left) and monolayer cultured hUCB iNSCs (right). (I) Morphology of homogeneous hUCB iNSCs cultured on PLO/FN-coated plates at passage 6. Scale bars in (E, F, H, I) and (G) are 250 μm and 100 μm, respectively.

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  Fig. 2 Characterization of hUCB iNSCs. (A) Immunocytochemistry analysis of NSC-specific marker proteins in hUCB iNSCs using antibodies against SOX2, PAX6, NESTIN, HMGA2, and KI67. Nuclei were counterstained with DAPI. Scale bars, 50 μm. (B) Relative expression levels of the neural stem cell-specific markers were measured through qRT-PCR in two lines of hUCB iNSCs and H9-NSCs. (C) Relative expression levels of the pluripotent stem cell-specific markers were measured through qRT-PCR in human embryonic stem cell (hESC), hUCB CD34+ cells, two lines of hUCB iNSCs and H9-NSCs. Error bars denote the standard deviation of triplicate reactions.

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  Fig. 3 In vitro differentiation of hUCB iNSCs. (A~C) Immunocytochemical analysis of hUCB iNSCs after differentiation into three major cell types: neurons (NF) (A); astrocytes (GFAP) (B); and oligodendrocytes (OLIG2 and O4) (C). Scale bars, 50 μm.

Reference

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