Mar 16, 2020
Human embryonic stem cells differentiation into oligodendrocyte lineage cells
- Paria Pooyan1,2,3,
- Hossein Baharvand2,3,4,
- Mohammad Javan3,5,
- Ghasem Hosseini Salekdeh1,6
- 1Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran;
- 2Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran;
- 3Department of Brain and Cognitive Science, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran;
- 4Department of Developmental Biology, University of Science and Culture, Tehran, Iran;
- 5Department of Physiology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran;
- 6Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran
Protocol Citation: Paria Pooyan, Hossein Baharvand, Mohammad Javan, Ghasem Hosseini Salekdeh 2020. Human embryonic stem cells differentiation into oligodendrocyte lineage cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bddni25e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We used this protocol in our group and now we are trying to share our findings with the enthusiastic scientists. We are also interested to improve the protocol.
In fact, this protocol is the customized version of Dr. Douvaras and Dr. Fossati's published protocol (Nature Protocols, 2015).
Created: March 08, 2020
Last Modified: March 16, 2020
Protocol Integer ID: 33934
Keywords: human embryonic stem cell, neural stem cell, neural progenitor cell, pre-oligodendrocyte progenitor cell, oligodendrocyte progenitor cell, oligodendrocyte,
Abstract
Oligodendrocyte (OL) lineage cell generation from human embryonic stem cell line Royan H6 (RH6) started by dual inhibition of SMAD signaling. Therefore, within eight days, adherent NANOG+RH6 cells (B) differentiated into SOX1+ neural stem cells (C). Next, the embryonic ventral spinal cord environment (pMN domain) was mimicked by the application of two morphogens, RA and SAG and the OLIG2+ neural progenitor cells were generated on day 12 (D) of differentiation. Further, oligodendrocyte progenitor cell commitment was achieved on day 20, when the pre-oligodendrocyte progenitor cell (pre-OPC) aggregates were positively stained by NKX2.2+ (E). From day 20 to day 80, supportive reagents for pre-OPCs maturation into PDGFRA+ OPCs (F), and their further expansion and maturation toward oligodendrocyte producing OPCs were added to the culture medium. Finally, on day 80 the growth-factors were withdrawn from the culture medium and OPCs were differentiated into MBP+ OLs (G).
References:
- Baharvand H, Ashtiani SK, Taee A, Massumi M, Valojerdi MR, Yazdi PE, et al. Generation of new human embryonic stem cell lines with diploid and triploid karyotypes. Dev Growth Differ. 2006;48:117–28.
- Douvaras P, Wang J, Zimmer M, Hanchuk S, O’Bara MA, Sadiq S, et al. Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells. Stem Cell Reports [Internet]. The Authors; 2014;3:250–9. Available from: http://dx.doi.org/10.1016/j.stemcr.2014.06.012
- Douvaras P, Fossati V. Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells. Nat Protoc [Internet]. 2015;10:1143–54. Available from: http://dx.doi.org/10.1038/nprot.2015.075
- Piao J, Major T, Auyeung G, Policarpio E, Menon J, Droms L, et al. Human embryonic stem cell-derived oligodendrocyte progenitors remyelinate the brain and rescue behavioral deficits following radiation. Cell Stem Cell [Internet]. Elsevier Inc.; 2015;16:198–210. Available from: http://dx.doi.org/10.1016/j.stem.2015.01.004
Materials
| Media | Components | Provider | Final Conc. | |
| NI medium | DMEM/F12 | Life Technologies | ||
| Knock-Out Serum❊ | Life Technologies | 5% | ||
| MEM Non-Essential Amino Acids (100X) | Life Technologies | 1X | ||
| GlutaMAX-I (100X) | Life Technologies | 1X | ||
| Penicillin-Streptomycin (100X) | Life Technologies | 1X | ||
| 2-Mercaptoethanol (1000X) | Life Technologies | 1X | ||
| Insulin-Transferrin-Selenium (100X) | Life Technologies | 1X | ||
| N-2 Supplement (100X) | Life Technologies | 1X | ||
| SB431542 | Stemgent | 10 µM | ||
| LDN193189 | Stemgent | 250 nM | ||
| All Trans-Retinoic Acid | Sigma | 100 nM | ||
| N2 medium | DMEM/F12 | Life Technologies | ||
| MEM Non-Essential Amino Acids (100X) | Life Technologies | 1X | ||
| GlutaMAX-I (100X) | Life Technologies | 1X | ||
| Penicillin-Streptomycin (100X) | Life Technologies | 1X | ||
| 2-Mercaptoethanol (1000X) | Life Technologies | 1X | ||
| Insulin-Transferrin-Selenium (100X) | Life Technologies | 1X | ||
| N-2 Supplement (100X) | Life Technologies | 1X | ||
| All Trans-Retinoic Acid | Sigma | 100 nM | ||
| Agonist of Sonic Hedgehog (SAG) | Stemgent | 1 µM | ||
| N2B27 medium | DMEM/F12 | Life Technologies | ||
| MEM Non-Essential Amino Acids (100X) | Life Technologies | 1X | ||
| GlutaMAX-I (100X) | Life Technologies | 1X | ||
| Penicillin-Streptomycin (100X) | Life Technologies | 1X | ||
| 2-Mercaptoethanol (1000X) | Life Technologies | 1X | ||
| Insulin-Transferrin-Selenium (100X) | Life Technologies | 1X | ||
| N-2 Supplement (100X) | Life Technologies | 1X | ||
| B-27 Supplement without Vitamin A (50X) | Life Technologies | 1X | ||
| All Trans-Retinoic Acid | Sigma | 100 nM | ||
| SAG | Stemgent | 1 µM | ||
| PDGF medium | DMEM/F12 | Life Technologies | ||
| MEM Non-Essential Amino Acids (100X) | Life Technologies | 1X | ||
| GlutaMAX-I (100X) | Life Technologies | 1X | ||
| Penicillin-Streptomycin (100X) | Life Technologies | 1X | ||
| 2-Mercaptoethanol (1000X) | Life Technologies | 1X | ||
| N-2 Supplement (100X) | Life Technologies | 1X | ||
| B-27 Supplement without Vitamin A (50X) | Life Technologies | 1X | ||
| Insulin | Sigma | 25 µg/ml | ||
| PDGF-AA | Sigma | 10 ng/ml | ||
| HGF | Sigma | 5 ng/ml | ||
| IGF-1 | Sigma | 10 ng/ml | ||
| NT3 | Royan Biotech | 10 ng/ml | ||
| T3 | Sigma | 60 ng/ml | ||
| Biotin | Sigma | 100ng/ml | ||
| cAMP | Sigma | 1µM | ||
| Glial medium | DMEM/F12 | Life Technologies | ||
| MEM Non-Essential Amino Acids (100X) | Life Technologies | 1X | ||
| GlutaMAX-I (100X) | Life Technologies | 1X | ||
| Penicillin-Streptomycin (100X) | Life Technologies | 1X | ||
| 2-Mercaptoethanol (1000X) | Life Technologies | 1X | ||
| N-2 Supplement (100X) | Life Technologies | 1X | ||
| B-27 Supplement without Vitamin A (50X) | Life Technologies | 1X | ||
| Insulin | Sigma | 25 µg/ml | ||
| HEPES | Sigma | 10 mM | ||
| T3 | Sigma | 60 ng/ml | ||
| Biotin | Sigma | 100ng/ml | ||
| cAMP | Sigma | 1µM | ||
| AA | Sigma | 20 µg/ml |
❊ From day four of differentiation, Knock-Out serum was gradually replaced by N-2 supplement (25%, 50%, 75%) as described previously.
6 ×106 RH6 cells were plated on Engelbreth-Holm-Swarm mouse sarcoma ECM (sigma) coated 6cm cell culture plates on day -1.
On day 0, the FFC medium (containing 20% KSR and 100 ng/ml bFGF) was replaced by 4 ml NI medium. NI medium was refreshed every day.
On day 8, the NI medium was replaced with N2 medium and the medium was refreshed every day.
From day 4, 5% KSR was gradually withdrawn from the NI medium. Respectively, 3.75%, 2.5%, 1.25% and 0% KSR was applied in the NI medium on day 4, 5, 6 and 7.
On day 12, cells’ attachment was loosened by PBS- for 10 min. Then PBS- was replaced with the N2B27 medium and cells were detached by braking their layer via the tip of the p1000 pipette. The cell clumps were pipetted gently with a p1000 pipette, for 5 times.
Detached cells were transferred into the 6 cm untreated plates to form aggregates and from day 14, two-thirds of the N2B27 medium was refreshed every day. The debris and dead cells were removed through the medium refreshment. Meanwhile, the aggregates that stuck to each other were broken apart by gentle pipetting (with the p1000 pipette).
On day 20, two-thirds of the N2B27 medium was replaced with PDGF medium. Two-thirds of the PDGF medium was refreshed every day until day 80.
On day 29, new 6 cm culture dishes were coated for overnight by 50 μg/ml poly-l-ornithine in dH2O, at 37 ℃. Next, they were coated by 20 μg/ml laminin in PBS for 12 h at 37 ℃.
On day 30, golden or brown aggregates (with a dark center) were plated on poly-l-ornithine/laminin coated dishes, with a p200 pipette (50 spheres per each 6 cm culture plate). The daily refreshment of the two-third of the PDGF medium was performed gently until day80.
FFirst on day 65 and then on day 75, cells and aggregates were re-plated on newly poly-l-ornithine/laminin coated dishes to achieve a more homogenous OPC population on day 80. PBS- and the p200 pipette were used for detachment and replacement.
On day 80, the PDGF medium was replaced by the Glial medium. Two-thirds of the medium was refreshed every two to three days, until day 120.