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Fast and efficient neural conversion of human hematopoietic cells.

Castaño J, Menendez P, Bruzos-Cidon C, Straccia M, Sousa A, Zabaleta L, Vazquez N, Zubiarrain A, Sonntag KC, Ugedo L, Carvajal-Vergara X, Canals JM, Torrecilla M, Sanchez-Pernaute R, Giorgetti A - Stem Cell Reports (2014)

Bottom Line: SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs.However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation.Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency.

View Article: PubMed Central - PubMed

Affiliation: Josep Carreras Leukemia Research Institute, Cell Therapy Program of the University of Barcelona, Barcelona 08036, Spain.

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Characterization iNCs Derived from CD133-Positive CB Cells(A) Quantification of expression levels by qRT-PCR of a panel of early, immature, and mature neuronal markers in CD133-positive CB cells and CB-iNCs at 15, 30, and 45 days of neural induction and propagation. Data are represented as mean ± SD (n = 3 independent experiments). Expression of these genes was not detected in untransduced CD133-positive CB cells.(B) Representative phase-contrast live image (a) and immunostaining (b) of CB-iNCs at passage 5 cultured on matrigel in the presence of FGF2. CB-iNCs acquired a neural epithelial-like morphology and expressed early neural progenitor markers such as NESTIN and SOX2. The scale bar is as indicated in the panels.(C) qRT-PCR analysis showing transcriptional changes in the CB-iNCs propagated on matrigel in the presence of FGF2 for five passages relative to CB-iNCs at day 30 of neural induction. Data are represented as mean ± SD (n = 3 independent experiments).(D) SeV vectors analyzed by qRT-PCR after 20 passages before and after temperature treatment (39°C)-mediated silencing. Data are represented as mean ± SD (n = 3 independent experiments).See also Figure S2.
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fig2: Characterization iNCs Derived from CD133-Positive CB Cells(A) Quantification of expression levels by qRT-PCR of a panel of early, immature, and mature neuronal markers in CD133-positive CB cells and CB-iNCs at 15, 30, and 45 days of neural induction and propagation. Data are represented as mean ± SD (n = 3 independent experiments). Expression of these genes was not detected in untransduced CD133-positive CB cells.(B) Representative phase-contrast live image (a) and immunostaining (b) of CB-iNCs at passage 5 cultured on matrigel in the presence of FGF2. CB-iNCs acquired a neural epithelial-like morphology and expressed early neural progenitor markers such as NESTIN and SOX2. The scale bar is as indicated in the panels.(C) qRT-PCR analysis showing transcriptional changes in the CB-iNCs propagated on matrigel in the presence of FGF2 for five passages relative to CB-iNCs at day 30 of neural induction. Data are represented as mean ± SD (n = 3 independent experiments).(D) SeV vectors analyzed by qRT-PCR after 20 passages before and after temperature treatment (39°C)-mediated silencing. Data are represented as mean ± SD (n = 3 independent experiments).See also Figure S2.

Mentions: A more extensive quantitative RT-PCR (qRT-PCR) analysis of CB-iNCs confirmed a remarkable upregulation of TUJ1, doublecortin (DCX), and MAP2 already at 15 days after neural induction, whereas PAX6 and early neural markers (MUSASHI, SOX1, and NESTIN) were present at lower levels (Figure 2A). While recent work identified MASH1 as the most important driver of neural conversion in fibroblasts (Chanda et al., 2014), we observed only marginal induction of MASH1 expression in CB-iNCs (Figure 2A). In contrast, BRN2 was progressively activated in CB-iNCs during the neural conversion (Figure 2A), suggesting an involvement of BRN2 in this process, in line with other recent reports (Lujan et al., 2012; Zou et al., 2014).


Fast and efficient neural conversion of human hematopoietic cells.

Castaño J, Menendez P, Bruzos-Cidon C, Straccia M, Sousa A, Zabaleta L, Vazquez N, Zubiarrain A, Sonntag KC, Ugedo L, Carvajal-Vergara X, Canals JM, Torrecilla M, Sanchez-Pernaute R, Giorgetti A - Stem Cell Reports (2014)

Characterization iNCs Derived from CD133-Positive CB Cells(A) Quantification of expression levels by qRT-PCR of a panel of early, immature, and mature neuronal markers in CD133-positive CB cells and CB-iNCs at 15, 30, and 45 days of neural induction and propagation. Data are represented as mean ± SD (n = 3 independent experiments). Expression of these genes was not detected in untransduced CD133-positive CB cells.(B) Representative phase-contrast live image (a) and immunostaining (b) of CB-iNCs at passage 5 cultured on matrigel in the presence of FGF2. CB-iNCs acquired a neural epithelial-like morphology and expressed early neural progenitor markers such as NESTIN and SOX2. The scale bar is as indicated in the panels.(C) qRT-PCR analysis showing transcriptional changes in the CB-iNCs propagated on matrigel in the presence of FGF2 for five passages relative to CB-iNCs at day 30 of neural induction. Data are represented as mean ± SD (n = 3 independent experiments).(D) SeV vectors analyzed by qRT-PCR after 20 passages before and after temperature treatment (39°C)-mediated silencing. Data are represented as mean ± SD (n = 3 independent experiments).See also Figure S2.
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fig2: Characterization iNCs Derived from CD133-Positive CB Cells(A) Quantification of expression levels by qRT-PCR of a panel of early, immature, and mature neuronal markers in CD133-positive CB cells and CB-iNCs at 15, 30, and 45 days of neural induction and propagation. Data are represented as mean ± SD (n = 3 independent experiments). Expression of these genes was not detected in untransduced CD133-positive CB cells.(B) Representative phase-contrast live image (a) and immunostaining (b) of CB-iNCs at passage 5 cultured on matrigel in the presence of FGF2. CB-iNCs acquired a neural epithelial-like morphology and expressed early neural progenitor markers such as NESTIN and SOX2. The scale bar is as indicated in the panels.(C) qRT-PCR analysis showing transcriptional changes in the CB-iNCs propagated on matrigel in the presence of FGF2 for five passages relative to CB-iNCs at day 30 of neural induction. Data are represented as mean ± SD (n = 3 independent experiments).(D) SeV vectors analyzed by qRT-PCR after 20 passages before and after temperature treatment (39°C)-mediated silencing. Data are represented as mean ± SD (n = 3 independent experiments).See also Figure S2.
Mentions: A more extensive quantitative RT-PCR (qRT-PCR) analysis of CB-iNCs confirmed a remarkable upregulation of TUJ1, doublecortin (DCX), and MAP2 already at 15 days after neural induction, whereas PAX6 and early neural markers (MUSASHI, SOX1, and NESTIN) were present at lower levels (Figure 2A). While recent work identified MASH1 as the most important driver of neural conversion in fibroblasts (Chanda et al., 2014), we observed only marginal induction of MASH1 expression in CB-iNCs (Figure 2A). In contrast, BRN2 was progressively activated in CB-iNCs during the neural conversion (Figure 2A), suggesting an involvement of BRN2 in this process, in line with other recent reports (Lujan et al., 2012; Zou et al., 2014).

Bottom Line: SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs.However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation.Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency.

View Article: PubMed Central - PubMed

Affiliation: Josep Carreras Leukemia Research Institute, Cell Therapy Program of the University of Barcelona, Barcelona 08036, Spain.

Show MeSH
Related in: MedlinePlus