Induction of myelinating oligodendrocytes in human cortical spheroids

A technology of oligodendrocytes, spheroids, applied in the field of induction of myelinating oligodendrocytes in human cortical spheroids

Pending Publication Date: 2021-03-09
CASE WESTERN RESERVE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, although single-cell analysis of cortical spheroids has identified transcriptional profiles suggesting the presence of oligodendrocyte progenitor cells (OPCs), and rare oligodendrocytes have been identified in isolates, there has been no Protocol demonstrates reproducible generation and maturation of oligodendrocytes, the myelinating glia of the central nervous system (CNS) and the third major cell type of neural origin

Method used

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  • Induction of myelinating oligodendrocytes in human cortical spheroids
  • Induction of myelinating oligodendrocytes in human cortical spheroids
  • Induction of myelinating oligodendrocytes in human cortical spheroids

Examples

Experimental program
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Effect test

Embodiment 1

[0087] Example 1 Production of oligocortical spheroids

[0088] An exemplary protocol is described herein to generate cortical spheroids derived from (human) pluripotent stem cells (hPSCs) comprising oligodendrocyte progenitor cells by timed exposure to defined oligodendrocyte lineage growth factors and hormones (OPC) and myelinating oligodendrocytes.

[0089] First, applicants generated and modeled "neurons" using an optimized version of the 50-day protocol (Pasca et al., Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat Methods 12, 671-678 (2015), incorporated herein by reference). Cortical spheroid". See Variations in Example 7.

[0090] Following initial neurocortical patterning, Applicants treated platelet-derived growth factor-AA (PDGF-AA) and insulin-like growth factor-1 (IGF-1) to drive expansion of the native OPC population (pp. 50-60 day = "week 9"), followed by thyroid hormone (T3) to induce oligodendrocyte differenti...

Embodiment 2

[0093] Example 2 Induction of OPCs and Oligodendrocytes

[0094] By the end of neurocortical patterning at week 8, neurocortical spheroids contained few cells in the oligodendrocyte lineage, as evidenced by minimal immunostaining for two canonical OPC transcription factors, OLIG2 and SOX10 ( Figures 6B-6C ). However, subsequent treatment of patterned spheroids with PDGF-AA and IGF-1 for 10 days resulted in a substantial increase in OPC numbers in oligocortical spheroids compared to age-matched untreated neurocortical spheroids ( Figures 6C-6E ).

[0095] By week 14, neurocortical spheroids had produced robust populations of neurons and astrocytes, but no oligodendrocytes ( Figure 1B ), whereas oligocortical spheroids (treated with PDGF-AA / IGF-1 from day 50-60 and T3 from day 60-70) reproducibly generated strong bonds in all three hPSC lines. Dendroglial populations, as demonstrated by immunofluorescence for proteolipoprotein 1 (PLP1), the most abundant oligodendrocyte me...

Embodiment 3

[0101] Example 3 Oligodendrocyte maturation and myelination

[0102] After the initial oligocortical pattern is formed, spheroids can be maintained in basal medium for weeks to months. Applicants analyzed neuronal diversity and oligodendrocyte maturation at 20 and 30 weeks ( Figure 2A ). The spheroids at week 20 appeared to be relatively immature. In addition to MYRF-positive oligodendrocytes, they contain large early deep neuronal populations marked by CTIP2 and separate smaller populations of late superficial neurons marked by SATB2 in which MYRF-positive oligodendrocytes Glial cells spread throughout the entire area ( Figure 2B , 8A). However, neuronal populations showed substantial overlap, consistent with ongoing migration of younger SATB2 cells through deep layers.

[0103] As oligodendrocytes mature, they expand the cellular process of tracing and myelinating adjacent axons. Although PLP1 expression was robust as early as week 14 in culture, PLP1 immunofluoresce...

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Abstract

The invention described herein provides a method for generating oligocortical spheroids from (human) pluripotent stem cells. The cortical spheroids so generated produces maturing oligodendrocytes thatcan, for example, myelinate axons, and model myelin disease and drug effects.

Description

[0001] References to related applications [0002] This international patent application claims the benefit of the filing dates of U.S. Provisional Patent Application 62 / 658,901, filed April 17, 2018, and U.S. Provisional Patent Application 62 / 700,472, filed July 19, 2018, each in its entirety Incorporated herein by reference, including any Figures and Sequence Listing. [0003] governmental support [0004] This invention was made with government support under NS093357, NS095280, GM007250, HD084167 and CA043703 awarded by the National Institutes of Health. The government has certain rights in this invention. Background technique [0005] Human corticogenesis is a complex process that requires the coordinated generation, migration and maturation of distinct cell populations. While many groups have generated oligodendrocytes through in vitro 2D culture and forced aggregation of differentiated neuronal cells, hPSC-derived cortical spheroids exploit an intrinsic differentiatio...

Claims

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Application Information

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IPC IPC(8): C12N5/079
CPCC12N5/0622C12N2501/395C12N2501/135C12N2501/105C12N2501/999C12N2503/02C12N2506/45C12N2513/00G01N33/5058
Inventor P.特萨尔M.马达范Z.内文
Owner CASE WESTERN RESERVE UNIV
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