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Methods for differentiating pluripotent stem cells in dynamic suspension culture

A technology of human pluripotent stem cells and dynamic suspension, which is applied in the field of cell biology, neuroectoderm and glial lineage cells, and can solve problems such as not easy to expand

Pending Publication Date: 2021-11-12
LINEAGE CELL THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, EB-based dual SMAD inhibition is not easily scalable for the generation of large numbers of target cells and leads to a greater degree of variability in the cells obtained, partly because static culture of EBs leads to the formation of aggregates of different sizes and requires Frequent grinding throughout (Crompton LA, Byrne ML, Taylor H, Kerrigan TL, Bru-Mercier G, Badger JL, Barbuti PA, Jo J, Tyler SJ, Allen SJ, Kunath T, Cho K, Caldwell MA. Stepwise , non-adherent differentiation of human pluripotent stem cells to generate basalforebrain cholinergic neurons via hedgehog signaling. 2013Stem Cell Res.11(3):1206-21)

Method used

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  • Methods for differentiating pluripotent stem cells in dynamic suspension culture
  • Methods for differentiating pluripotent stem cells in dynamic suspension culture
  • Methods for differentiating pluripotent stem cells in dynamic suspension culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1 - Culture and Expansion of Undifferentiated Human Embryonic Stem Cells

[0112] From the H1 line (WA01; Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998 Nov 6; 282(5391): 1145- 7) Working Cell Bank (WCB) generated from undifferentiated human embryonic stem cells (uhESC) in recombinant human laminin-521 (Corning #354224) coated, tissue culture treated polystyrene 225cm 2 Complete mTeSR on culture flasks (Corning #431082) TM -1 medium (StemCell Technologies #85850). Complete media changes daily until cells reach approximately 80-90% confluency, then use ReLeSR TM Reagents (Stem Cell Technologies #05872) were used to passage uhESCs. Will ReLeSR TM Lifted uhESC cells were seeded onto fresh laminin-521-coated 225 cm 2 flasks, and resume daily medium changes two days after inoculation. Cultured uhESCs from WCBs were expanded in this way for 2 to 5...

Embodiment 2

[0113] Example 2 - Method for Differentiating Human Embryonic Stem Cells into Neuroectodermal Progenitor Cells in Dynamic Suspension Culture

[0114] Day 1 : Expanded uhESCs (approximately 90% confluent) were isolated and cultured with (Stem Cell Technologies #07920) disaggregates to form a single cell suspension, allowing accurate cell counts and uniform seeding density. The depolymerized uhESCs were then plated at 1x10 6 A concentration of viable cells / mL was inoculated into a PBS-0.1 or PBS-0.5 microbioreactor system (PBS Biotech) (set to rotate at 35RPM or 25RPM, respectively (Day -1)) for dynamic suspension culture. Cells were seeded in Glial Progenitor Cell Medium (GPM; prepared with 2% B27 supplement (Gibco cat. no. 17504-044) and 0.04 μg / ml triiodo-thyronine (Sigma cat. no. T5516-1MG) DMEM / F12 (Gibco Cat. No. 10565-018)) and undifferentiated hESC medium (as in Example 1) supplemented with 10 μM Rho Kinase Inhibitor (RI, Tocris Cat. No. 1254) to support cell surviv...

Embodiment 3

[0118] Example 3 - Method for Differentiating Human Embryonic Stem Cells into Glial Lineage Cells in Dynamic Suspension Culture

[0119] Differentiation of uhESCs to neuroectoderm / neural progenitor cells (days 0-6) was performed as described in Example 2. On day 7, the differentiation medium was supplemented with 20 ng / mL human basic fibroblast growth factor (hbFGF, ThermoFisher, catalog number PHG0263), 10 ng / mL epidermal growth factor (EGF, Thermo Fisher, cat. ) and 10 μM RI of GPM to initiate differentiation into glial progenitor cells. For the next two weeks (days 8-20), the cell aggregates were placed in dynamic suspension at 45 rpm (PBS-0.1 mini-bioreactor) or 32 RPM (PBS-0.5 mini-bioreactor) supplemented with 20 ng / GPM of mL bFGF and 10 ng / mL EGF was maintained, and the medium was replenished daily using gravity settling and 70-80% medium exchange. On day 14, 10 μM RI was also added to fresh medium.

[0120] A subset of differentiated cells was harvested at day 21 o...

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Abstract

Methods for differentiating pluripotent stem cells to neuroectoderm in dynamic suspension culture using small molecule or protein inhibitors of TGF[beta] / Activin / Nodal signaling and BMP signaling are provided. Also provided are method and protocols for differentiating pluripotent stem cells such as human embryonic stem cells first to neuroectoderm, then further to glial progenitor cells, and further to oligodendrocyte progenitor cells (OPCs), and compositions obtained thereby. The methods of the present disclosure reproducibly produce 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

technical field [0001] The present disclosure relates to the fields of cell biology and neuroectodermal and glial lineage cells (eg, oligodendrocyte progenitor cells). More specifically, the present disclosure relates to novel methods for differentiating pluripotent stem cells into neuroectoderm in dynamic suspension culture using small molecule or protein inhibitors of TGFβ / Activin / Nodal signaling and BMP signaling. The present disclosure further provides novel methods for differentiating pluripotent stem cells (eg, human embryonic stem cells) first into neuroectoderm, then further into glial progenitor cells, and further into oligodendrocyte progenitor cells. The present disclosure further relates to neuroectodermal cells, glial progenitor cells and oligodendrocyte progenitor cells expressing one or more markers produced by the methods according to the present invention. Background technique [0002] Oligodendrocyte progenitor cells (OPCs) are a subset of glial cells in t...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12N5/079
CPCC12N5/0622C12N2506/02C12N2533/52C12N2501/375C12N2501/727C12N2501/15C12N2501/16C12N2501/155C12N2501/385C12N2500/38C12N2501/115C12N2501/11C12N5/0068C12N2500/46C12N2506/03C12N2506/45C12N2501/165C12N2501/135C12N2501/41
Inventor R·R·奈尔S·凯泽A·S·帕里克U·肖卡特-蒙塔兹E·M·怀特利N·C·曼利C·R·哈尔贝施塔特
Owner LINEAGE CELL THERAPEUTICS INC
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