Method for highly efficient conversion of human stem cells to lineage-specific neurons

a human stem cell and lineage-specific technology, applied in the field of stem cells, can solve the problems of lack of specificity and efficiency of strategies, and achieve the effects of high efficiency of human scs conversion, rapid and highly efficient lineage-specific neuronal conversion, and high application valu

Inactive Publication Date: 2016-10-13
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE +1
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  • Abstract
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Benefits of technology

[0006]Cell replacement therapy using human stem cells (SCs) holds great promise for treating neurological diseases in which neuronal loss results in cognitive, extrapyramidal, and / or motor dysfunction. Optimized differentiation strategies are essential to differentiate human stem cells into lineage-specific neuronal progenies in sufficient numbers and purity for transplantation or disease modeling. As described herein, we have established a strategy for differentiating human pluripotent or multipotent SCs into lineage-specific and functional neurons. We found a defined transcription factor that can induce the highly efficient conversion of human SCs to lineage-specific neurons (e.g., dopaminergic (DA) neurons). This strategy can generate dopaminergic neurons with >80% purity. The established strategy is novel and highly applicable for disease modeling and cell replacement therapy for several neurological disorders, including but not limited to Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis and hearing loss.
[0007]Current differentiation strategies for converting human SCs to neurons act primarily on cell surface receptors or intracellular signaling proteins to alter the level of multiple downstream transcription factors and in turn to change the gene expression profile and cell fate specification. These strategies lack specificity and efficiency because they activate multiple signaling cascades and transcription factors, only a fraction of which are optimal and necessary to drive lineage-specific neuronal differentiation. In certain embodiments, our differentiation strategy uses a single transcription factor Atoh1 to induce highly specific neuronal differentiation signaling, resulting in rapid and highly efficient lineage-specific neuronal conversion. In other embodiments, the strategy may include NeuroD1 and / or Neurogenin 2. Our differentiation strategy saves the time for generating lineage-specific neurons from human SCs by at least 50%. It also generates lineage-specific neurons with >80% purity, significantly higher than the purity (10-40%) normally achieved by other methods.

Problems solved by technology

These strategies lack specificity and efficiency because they activate multiple signaling cascades and transcription factors, only a fraction of which are optimal and necessary to drive lineage-specific neuronal differentiation.

Method used

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  • Method for highly efficient conversion of human stem cells to lineage-specific neurons
  • Method for highly efficient conversion of human stem cells to lineage-specific neurons
  • Method for highly efficient conversion of human stem cells to lineage-specific neurons

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example 1

Proneural Transcription Factor Atoh1 Drives Highly Efficient Differentiation of Human Pluripotent Stems Cells into Dopaminergic Neurons

Materials and Methods

[0041]Cell Culture.

[0042]Human H1 ESC line was obtained from WiCell Research Resources (WiCell, WI). Human iPSC line ND27760 (passage 25-30) was derived from human skin fibroblasts from a PD patient with a SNCA triplication that were obtained from the Coriell Cell Repositories. Cell reprogramming was performed using non-integrating 4 factor (SOX2 / OCT4 / KLF4 / MYC) Sendai virus system (CytoTune-iPS Reprogramming Kit, Life Technologies). The pluripotency of this iPSC line has been characterized by immunocytochemistry for pluripotent cell markers (NANOG, OCT4, TRA-1-60 and SSEA-3) and embryoid body differentiation. Human ESCs and iPSCs were maintained as feeder-free cultures in Essential 8 medium (Life Technologies) or mTESR1 medium (Stemcell Technologies) in 5% CO2 / 95% air condition at 37° C., and were passaged using dispase (Life Tec...

example 2

Atoh1-Mediated Differentiation of Human ESCs and NSCs into DA Neurons

[0092]Atoh1-ESCs were differentiated following the protocol as shown in FIG. 4A and Table 2. Atoh1-induced neuron cultures at Day 36 co-expressed neuronal and DA neuron markers, β-tubulin III (TUJ1) and tyrosine hydroxylase (TH), respectively (FIG. 12A). The Atoh1 induction protocol yielded DA neurons from human ESCs with 84±9% purity as determined by the percentage of TUJ+ / TH+ cells (FIG. 12A). Atoh1-NSCs were differentiated following the protocol as shown in FIG. 4A and Table 3. Atoh1-induced neuron cultures at Day 36 co-expressed neuronal marker (β-tubulin III, TUJ1) and DA neuron marker (tyrosine hydroxylase, TH) (FIG. 12B). The Atoh1 induction protocol yielded DA neurons from human NSCs with 82±8% purity as determined by the percentage of TUJ+ / TH+ cells (FIG. 12B).

TABLE 2DA neuron differentiation media used in Atoh1-induced protocol as shown in FIG. 4ADayMediumAdditives0E8Y-276321E6 (75%) + N2 (25%)Doxycyline / ...

example 3

Atoh1-Mediated Differentiation of Human iPSCs into Dopaminergic (DA) Neurons

[0093]As shown in FIG. 4A, we established protocols for differentiating human iPSCs into DA neurons by Atoh1 and other additives (sonic hedgehog and FGF-8b). The recipes for cell culture media are listed in Table 2. Cells were plated (4×104 cells per cm2) on matrigel (BD) in Essential 8 Medium (Life Technologies) with ROCK inhibitor (Y-27632, Stemgent). From Day 1 to Day 7, cell culture medium was changed every day. From Day 8 to Day 36, half of the cell culture medium was changed every 3-4 days. At day 36 of differentiation, these neurons co-expressed neuronal marker (β-tubulin III, TUJ1) and DA neuron marker (tyrosine hydroxylase, TH), the rate-limiting enzyme in the synthesis of dopamine (FIG. 4C). The Atoh1 induction protocol yielded DA neurons with 89±6% purity as determined by the percentage of TUJ+ / TH+ cells (FIG. 4D). Moreover, the Atoh1-induced DA neurons also co-expressed other midbrain DA neuron m...

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Abstract

The present invention relates to the field of stem cells. More specifically, the present invention provides methods and compositions useful for the highly efficient conversion of human stem cells to lineage-specific neurons. In a specific embodiment, a method of inducing differentiation of human stem cells into dopaminergic (DA) neurons comprises the steps of (a) transfecting human stem cells with a lentiviral vector encoding Atoh1, wherein the vector is Dox inducible; and (b) growing the transfected cells in culture in the presence of Dox, Sonic Hedgehog (SHH) and FGF-8b until DA neurons are induced.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 61 / 911,238, filed Dec. 3, 2013, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0002]The present invention relates to the field of stem cells. More specifically, the present invention provides methods and compositions useful for the highly efficient conversion of human stem cells to lineage-specific neurons.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY[0003]This application contains a sequence listing. It has been submitted electronically via EFS-Web as an ASCII text file entitled “P12596-02_ST25.txt.” The sequence listing is 21,808 bytes in size, and was created on Dec. 3, 2014. It is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION[0004]Optimized differentiation strategies are essential for differentiating human stem cells (SCs) into lineage-specific neuronal progenies in sufficient numbe...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/0793C12N15/85C12N15/86A61K35/30
CPCC12N5/0619A61K35/30C12N15/85C12N15/86C12N2740/15043C12N2506/08C12N2501/115C12N2501/41C12N2506/02C12N2506/45C12N2510/00C12N2501/60A61P25/00C12N2500/38C12N2501/01C12N2501/119C12N2501/13C12N2501/15C12N2533/32C12N2533/52C12N2799/027
Inventor YING, MINGYAOLATERRA, JOHN
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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