Open Access

Comparison of methods for deriving neural progenitor cells from nonhuman primate embryonic stem cells

Apitsada Khlongkhlaeo
Apitsada Khlongkhlaeo
, 
Richard L. Carter
Richard L. Carter
, 
Rangsun Parnpai
Rangsun Parnpai
 and 
Anthony W.S. Chan
Anthony W.S. Chan
   | Mar 31, 2017
Asian Biomedicine's Cover Image
About this article
Previous Article
Next Article
Cite
Article Category: Original article
Published Online: Mar 31, 2017
Page range: 423 - 433
DOI: https://doi.org/10.5372/1905-7415.1005.505
Keywords
© 2016 Apitsada Khlongkhlaeo, Richard L. Carter, Rangsun Parnpai, Anthony W.S. Chan
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Induction of rhesus monkey (Macaca mulatta) embryonic stem cells (rhESCs) to become neural progenitors in an adherent monolayer without feeder cells. (A) Schematic for the monolayer cell induction method. (B) rhESC colony cultured on a mouse embryonic fibroblast feeder layer before transfer to feeder free conditions. (C) rhESC cultured in neural induction medium after 4 passages without feeders. (D) Neural rosette-like structure (arrows) formed at day 8 (D8) of differentiation. (E) rhESCs derived neural progenitor cells.
Induction of rhesus monkey (Macaca mulatta) embryonic stem cells (rhESCs) to become neural progenitors in an adherent monolayer without feeder cells. (A) Schematic for the monolayer cell induction method. (B) rhESC colony cultured on a mouse embryonic fibroblast feeder layer before transfer to feeder free conditions. (C) rhESC cultured in neural induction medium after 4 passages without feeders. (D) Neural rosette-like structure (arrows) formed at day 8 (D8) of differentiation. (E) rhESCs derived neural progenitor cells.

Figure 2

Derivation of neural progenitor cells from rhesus monkey (Macaca mulatta) embryonic stem cells (rhESCs) using the embryoid body (EB) method. (A) Schematic diagram of EB induction protocol. (B) rhESC derived EBs in suspension. (C) Adherent culture of EBs in an induction medium with small neural rosette-like structures (arrows). (D) The rosette-like structures were isolated and replated followed by second round of selection neural rosette.
Derivation of neural progenitor cells from rhesus monkey (Macaca mulatta) embryonic stem cells (rhESCs) using the embryoid body (EB) method. (A) Schematic diagram of EB induction protocol. (B) rhESC derived EBs in suspension. (C) Adherent culture of EBs in an induction medium with small neural rosette-like structures (arrows). (D) The rosette-like structures were isolated and replated followed by second round of selection neural rosette.

Figure 3

Characterization of rhesus monkey (Macaca mulatta) embryonic stem cells (rhESC)-derived neural progenitor cells (NPCs). (A) After derivation, the cells from both methods of derivation showed NPC morphology and stained for NPC markers; Sox2, Musashi, and nestin. (B) The expression of NPC-related genes was measured by real-time polymerase chain reactions. Data are the mean of fold change relative to rhESC gene expression ± SEM (samples run in duplicate, n = 3, P < 0.05). (C) Flow cytometry analysis of rhESC-derived NPCs with 99.1% and 99.5% were Pax6-positive and 97.0% and 99.4% were nestin-positive for feeder-free and embryoid body derived NPCs, respectively. The x-axes represent the fluorescent intensity and y-axes show the number of cells.
Characterization of rhesus monkey (Macaca mulatta) embryonic stem cells (rhESC)-derived neural progenitor cells (NPCs). (A) After derivation, the cells from both methods of derivation showed NPC morphology and stained for NPC markers; Sox2, Musashi, and nestin. (B) The expression of NPC-related genes was measured by real-time polymerase chain reactions. Data are the mean of fold change relative to rhESC gene expression ± SEM (samples run in duplicate, n = 3, P < 0.05). (C) Flow cytometry analysis of rhESC-derived NPCs with 99.1% and 99.5% were Pax6-positive and 97.0% and 99.4% were nestin-positive for feeder-free and embryoid body derived NPCs, respectively. The x-axes represent the fluorescent intensity and y-axes show the number of cells.

Figure 4

Differentiation of rhesus monkey (Macaca mulatta) neural progenitor cells (rhNPCs) into neural cells. (A) rhNPCs from feeder free-based and (B) EB-based methods showed highly branched morphology and elongated spindle processes, consistent with neuronal morphology. (C-D) Immunoreactivity revealed MAP2-positive cells in neural cells differentiated from feeder free and EB derived NPCs. (E) Quantitative real time polymerase chain reactions revealed the expression of MAP2 in neural cells derived from the 2 groups (n = 3, P < 0.05).
Differentiation of rhesus monkey (Macaca mulatta) neural progenitor cells (rhNPCs) into neural cells. (A) rhNPCs from feeder free-based and (B) EB-based methods showed highly branched morphology and elongated spindle processes, consistent with neuronal morphology. (C-D) Immunoreactivity revealed MAP2-positive cells in neural cells differentiated from feeder free and EB derived NPCs. (E) Quantitative real time polymerase chain reactions revealed the expression of MAP2 in neural cells derived from the 2 groups (n = 3, P < 0.05).

Figure 5

Expression of nonneural lineage and neural lineage markers. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that 3 independent rhesus neural progenitor cell lines derived from feeder-free and embryoid body (EB) methods showed similar gene expression patterns for (A) nonneural lineage and (B) expression of astrocyte markers, GFAP and S100β. (C) The expression of GFAP was confirmed by immunoreactivity and on neural cells differentiated from feeder-free derived NPCs. (D) Nuclear receptor tailless (TLX) expression was measured by qRT-PCR in neural cells differentiated from feeder-free derived NPCs (n = 3, P < 0.05).
Expression of nonneural lineage and neural lineage markers. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that 3 independent rhesus neural progenitor cell lines derived from feeder-free and embryoid body (EB) methods showed similar gene expression patterns for (A) nonneural lineage and (B) expression of astrocyte markers, GFAP and S100β. (C) The expression of GFAP was confirmed by immunoreactivity and on neural cells differentiated from feeder-free derived NPCs. (D) Nuclear receptor tailless (TLX) expression was measured by qRT-PCR in neural cells differentiated from feeder-free derived NPCs (n = 3, P < 0.05).
eISSN:
1875-855X
Language:
English
Publication timeframe:
6 times per year
Journal Subjects:
Medicine, Assistive Professions, Nursing, Basic Medical Science, other, Clinical Medicine
Journal RSS Feed