Dorsally-derived oligodendrocyte progenitor cells from human pluripotent stem cells

Abstract

Methods for differentiating human pluripotent stem cells to dorsal neuroectoderm progenitors and further to glial progenitor cells and oligodendrocyte progenitor cells (OPCs) using inhibitors of BMP signaling and MAPK / ERK signaling are provided. Also provided are cells and cellular compositions obtained by such methods, and uses of such cells. Further provided are methods and protocols for efficiently differentiating human pluripotent stem cells to OPCs in the absence of the ventralizing morphogen SHH or a SHH signaling activator. The methods of the present disclosure reproducibly produce dorsal neuroectoderm progenitor cells by day 7 of the differentiation process, glial progenitor cells by day 21 of the differentiation process and OPCs by day 42 of the differentiation process.

Description

[0001] Related application

[0002] The present application claims priority to US Provisional Application No. 62 / 796, 077, filed on Jan. 23, 2019, which is incorporated herein by reference. Technical field

[0003] The present disclosure relates to a neurosteilic progenitor cell having a neurodegelery having a phenotype having a backnirotropy of a veneer, and then further differentiates into a neuroperic progenitor cell, and further differentiation is a small gelatin progenitor cell. new method. Cells and cell compositions obtained by such methods, as well as use of such cells. The present disclosure further relates to cells expressing one or more markers by the method according to the invention. Background technique

[0004] Less epithelial progenitor cells (OPC) are subtypes of geriroid cells in the central nervous system (CNS), which appears in the brain and spinal ventricle, and migrates to the entire development of maturity for less epitonal cells. CNS in. Mature else gel cel...

Examples

Embodiment 1

[0138] Example 1 - Cultivation and amplification of undifferentiated human embryonic stem cells

[0139] From the H1 Series (WA01; Thomson Ja, ITSKOVITZ-Eldor J, SHAPIRO SS, WAKNITZ MA, SWIERGIEL JJ, MARSHALL VS, JONES JM.EMBRYONIC STEM CELL LINES DERIVED ": 1145- 7) Unexplified human embryonic stem cells (UHESC) of the generated working cell library (UHESC) in recombinant human adhesion protein-521 (RHLN-521, Corning # 354224) coated tissue cultured polystyrene T- 75 Culture Bottle (Corning # 431082) on complete MTESR TM The culture of -1 medium (STEM CELL TECHNOLOGIES # 85850). Replace the medium per day until the cells reached a conversion of about 80-90%, then use Reserv TM The reagent (STEM CELL TECHNOLOGIES # 05872) was subjected to UHESC. Relasr TM Lifted UHESC cells in the new RHLN-521 package 225cm 2 In the culture flask, the daily medium replacement is restored two days after inoculation. Differentiation as described in Example 2 Before the neurometry progenitor cells we...

Embodiment 2

[0140] Example 2 Method for differentiation of human embryonic stem cells into nerve pesticide progenitor cells with phenotype of back riropeus

[0141] The amplified UHESC was inoculated on the RHLN-521 coated container, and cultured until 40-70% of the conversion, at which time the origin was initiated.

[0142] Day 0-3: By removing MTESR TM -1 medium and added addition to 10 μM Mapk / Erk inhibitor PD0325901 (Pd; Sigma-Aldrich catalog number PZ0162), 2 μm BMP signaling inhibitor Dorsomorphin (Dorso; Sigma-Aldrich catalog number P5499) and 1 μM retinoic acid (RA Sigma-Aldrich catalog number R2625) of the neuropental progenitor cell culture medium (GPM; 2% B27 supplement (GIBCO catalog 17504-044) and 0.04 μg / ml triiodomethionyl epithan (Sigma-Aldrich) The DMEM / F12 (GIBCO directory number 10565-018) of catalog number T5516-1 mg is composed to start differentiation. This medium is supplemented daily.

[0143] Day 4-6: On day 4, the medium was switched to add 1 μM Ra and 150 μm ...

Embodiment 3

[0145] Example 3 - Method for differentiation of human embryonic stem cells into neurose mass spectrometry cells

[0146] Day 7-13: UHESC is carried out to differentiate to neurological pest progenitor cells (especially having back side phenotype) as described in Example 2. On day 7, use Tryple TM SELECT (Thermo Fisher, catalog number A12859-01) Raised cells, count, and in 2.7x10 4 Cell / cm 2 The inoculation density is supplemented with 20 ng / ml human alkaline fibroblast growth factor (HBFGF, THERMO FISHER, catalog number PHG0263), 10 ng / ml epidermal growth factor (EGF, THERMO FISHER, catalog number PHG0311) and 10 μM RhO kinase inhibitor (RI, TOCRIS directory number 1254) is inoculated into the RHLN-521 coated container. The medium was replaced by a medium with fresh GPM + HBFGF + EGF daily.

[0147] Day 14-21: On day 14, use Tryple TM SELECT raises cells, counts, resuspended in GPM + HBFGF + EGF + RI, and 1.83x10 6 The density of live cells / mL was re-inoculated into a dyn...

[0001] Related application

[0002] The present application claims priority to US Provisional Application No. 62 / 796, 077, filed on Jan. 23, 2019, which is incorporated herein by reference. Technical field

[0003] The present disclosure relates to a neurosteilic progenitor cell having a neurodegelery having a phenotype having a backnirotropy of a veneer, and then further differentiates into a neuroperic progenitor cell, and further differentiation is a small gelatin progenitor cell. new method. Cells and cell compositions obtained by such methods, as well as use of such cells. The present disclosure further relates to cells expressing one or more markers by the method according to the invention. Background technique

[0004] Less epithelial progenitor cells (OPC) are subtypes of geriroid cells in the central nervous system (CNS), which appears in the brain and spinal ventricle, and migrates to the entire development of maturity for less epitonal cells. CNS in. Mature else gel cel...

Examples

Embodiment 1

[0138] Example 1 - Cultivation and amplification of undifferentiated human embryonic stem cells

[0139] From the H1 Series (WA01; Thomson Ja, ITSKOVITZ-Eldor J, SHAPIRO SS, WAKNITZ MA, SWIERGIEL JJ, MARSHALL VS, JONES JM.EMBRYONIC STEM CELL LINES DERIVED ": 1145- 7) Unexplified human embryonic stem cells (UHESC) of the generated working cell library (UHESC) in recombinant human adhesion protein-521 (RHLN-521, Corning # 354224) coated tissue cultured polystyrene T- 75 Culture Bottle (Corning # 431082) on complete MTESR TM The culture of -1 medium (STEM CELL TECHNOLOGIES # 85850). Replace the medium per day until the cells reached a conversion of about 80-90%, then use Reserv TM The reagent (STEM CELL TECHNOLOGIES # 05872) was subjected to UHESC. Relasr TM Lifted UHESC cells in the new RHLN-521 package 225cm 2 In the culture flask, the daily medium replacement is restored two days after inoculation. Differentiation as described in Example 2 Before the neurometry progenitor cells we...

Embodiment 2

[0140] Example 2 Method for differentiation of human embryonic stem cells into nerve pesticide progenitor cells with phenotype of back riropeus

[0141] The amplified UHESC was inoculated on the RHLN-521 coated container, and cultured until 40-70% of the conversion, at which time the origin was initiated.

[0142] Day 0-3: By removing MTESR TM -1 medium and added addition to 10 μM Mapk / Erk inhibitor PD0325901 (Pd; Sigma-Aldrich catalog number PZ0162), 2 μm BMP signaling inhibitor Dorsomorphin (Dorso; Sigma-Aldrich catalog number P5499) and 1 μM retinoic acid (RA Sigma-Aldrich catalog number R2625) of the neuropental progenitor cell culture medium (GPM; 2% B27 supplement (GIBCO catalog 17504-044) and 0.04 μg / ml triiodomethionyl epithan (Sigma-Aldrich) The DMEM / F12 (GIBCO directory number 10565-018) of catalog number T5516-1 mg is composed to start differentiation. This medium is supplemented daily.

[0143] Day 4-6: On day 4, the medium was switched to add 1 μM Ra and 150 μm ...

Embodiment 3

[0145] Example 3 - Method for differentiation of human embryonic stem cells into neurose mass spectrometry cells

[0146] Day 7-13: UHESC is carried out to differentiate to neurological pest progenitor cells (especially having back side phenotype) as described in Example 2. On day 7, use Tryple TM SELECT (Thermo Fisher, catalog number A12859-01) Raised cells, count, and in 2.7x10 4 Cell / cm 2 The inoculation density is supplemented with 20 ng / ml human alkaline fibroblast growth factor (HBFGF, THERMO FISHER, catalog number PHG0263), 10 ng / ml epidermal growth factor (EGF, THERMO FISHER, catalog number PHG0311) and 10 μM RhO kinase inhibitor (RI, TOCRIS directory number 1254) is inoculated into the RHLN-521 coated container. The medium was replaced by a medium with fresh GPM + HBFGF + EGF daily.

[0147] Day 14-21: On day 14, use Tryple TM SELECT raises cells, counts, resuspended in GPM + HBFGF + EGF + RI, and 1.83x10 6 The density of live cells / mL was re-inoculated into a dyn...

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