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Dopaminergic neurons and proliferation-competent precursor cells for treating Parkinson's disease

a technology of dopaminergic neurons and precursor cells, applied in the field of dopaminergic neurons and proliferation-competent precursor cells for treating parkinson's disease, can solve the problems of affecting the normal functioning of the nervous system, the inability to reverse the damage of the disease, and the devastating consequences of the disease for those affected

Inactive Publication Date: 2005-02-24
ASTERIAS BIOTHERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes a system for efficiently producing neural cells from pluripotent cells, such as embryonic stem cells. The invention provides a population of cells with features characteristic of neural cells, such as neurons and glial cells, and can be used for drug testing and therapy to restore nervous system function. The invention also includes a method for optimizing differentiation protocols for producing neural cells with a desired phenotype. The cells can be used for screening compounds for neural cell toxicity or modulation, and can be formulated as a medicament for treating conditions affecting the nervous system. Overall, the invention provides a reliable and efficient way to produce neural cells for research and therapeutic purposes.

Problems solved by technology

Regardless of the age of presentation, the disease often has devastating consequences for those afflicted.
What makes afflictions of the nervous system so difficult to manage is the irreversibility of the damage often sustained.
But there is a severe shortage of suitable tissue.
Unfortunately, it has not been shown that progenitors isolated from neural tissue have sufficient replicative capacity to produce the number of cells necessary for human clinical therapy.

Method used

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  • Dopaminergic neurons and proliferation-competent precursor cells for treating Parkinson's disease
  • Dopaminergic neurons and proliferation-competent precursor cells for treating Parkinson's disease
  • Dopaminergic neurons and proliferation-competent precursor cells for treating Parkinson's disease

Examples

Experimental program
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example 1

NCAM-Positive Cells

This experiment focused on determining whether the human embryonic stem cells (hES) could undergo directed differentiation to NCAM-positive progenitor cells.

hES cells were harvested either from cultures supported by embryonic fibroblasts, or from feeder-free cultures, as described previously (AU 729377; WO 01 / 51616). Embryoid bodies were produced as follows. Confluent monolayer cultures of hES cells were harvested by incubating in 1 mg / mL collagenase for 5-20 min, following which the cells are scraped from the plate. The cells were then dissociated into clusters and plated in non-adherent cell culture plates (Costar) in a medium composed of 80% KO (“knockout”) DMEM (Gibco) and 20% non-heat-inactivated FBS (Hyclone), supplemented with 1% non-essential amino acids, 1 mM glutamine, 0.1 mM β-mercaptoethanol. The cells are seeded at a 1:1 or 1:2 ratio in 2 mL medium per well (6 well plate).

After 4-8 days in suspension, the EBs were plated intact in DMEM / F12 mediu...

example 2

A2B5-Positive Cells

Cells in this experiment were immunoselected for the surface marker A2B5. hES cells were induced to form EBs in 20% FBS. After 4 days in suspension, the EBs were plated onto fibronectin in DMEM / F12 with N2 and B27 supplemented with 10 ng / mL human EGF, 10 ng / mL human bFGF, 1 ng / mL human IGF-I, and 1 ng / mL human PDGF-AA. After 2-3 days in these conditions, 25-66% of the cells express A2B5. This population is enriched by magnetic bead sorting to 48-93% purity (Table 2).

TABLE 2Differentiation and Sorting Conditions for A2B5-positive CellsCells staininghESFactors used inpositively for NCAMCell Line used forDifferentiationBeforePositiveNegativeDifferentiationCultureType of SortsortsortsortH7 p32 667.004C F N I Pbead sort257710H1 p43 667.010C F N I Pbead sort62n / a50H1 p44 667.012C F N I Pbead sort568932H1 p46 667.020E P F Ibead sort2748 2H1 p47 667.032E P F Ibead sort579330H9 p40MG 667.038E P F Ibead sort669341H9 p42 667.041E P F Ibead sort2770 6

Factor abbreviations:...

example 3

Differentiation to Mature Neurons

To generate terminally differentiated neurons, the first stage of differentiation was induced by forming embryoid bodies in FBS medium with or without 10 μM retinoic acid (RA). After 4 days in suspension, embryoid bodies were plated onto fibronectin-coated plates in defined medium supplemented with 10 ng / mL human EGF, 10 ng / mL human bFGF, 1 ng / mL human PDGF-AA, and 1 ng / mL human IGF-1. The embryoid bodies adhered to the plates, and cells began to migrate onto the plastic, forming a monolayer.

After 3 days, many cells with neuronal morphology were observed. The neural precursors were identified as cells positive for BrdU incorporation, nestin staining, and the absence of lineage specific differentiation markers. Putative neuronal and glial progenitor cells were identified as positive for polysialylated NCAM and A2B5. Forty one to sixty percent of the cells expressed NCAM, and 20-66% expressed A2B5, as measured by flow cytometry. A subpopulation of ...

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Abstract

This disclosure provides improved methods for obtaining populations of neural progenitor cells and differentiated neurons from pluripotent stem cells. The technology can be used to produce progenitors that proliferate through at least 40 doublings, while maintaining the ability to differentiate into a variety of different neural phenotypes. Cell populations have been obtained that contain a high proportion of cells staining for tyrosine hydroxylase, which is a feature of dopaminergic neurons. The neural progenitors and terminally differentiated neurons of this invention can be generated in large quantities for use in drug screening and the treatment of clinically important neurological disorders, such as Parkinson's disease.

Description

BACKGROUND New research into the derivation and expansion of cell lines suitable for human administration promises to usher in a brave new world medical care. Devastating and previously intractable disease conditions may yield to the promise of regenerative medicine, providing that science continues to benefit from important new discoveries in the cell biology of neurons and neural precursor cells. Amongst the disease conditions in need of a clinical advance are those relating to neurological dysfunction. Near the top of the list is Parkinson's disease, an idiopathic, slowly progressive, degenerative disorder of the central nervous system, characterized by slow and decreased movement, muscular rigidity, resting tremor, and postural instability. The symptoms ensue from progressive deterioration of pigmented neurons in the substantia nigra, locus caeruleus, and other brain stem dopaminergic cells, causing a depletion of the neurotransmifter dopamine. Parkinson's disease is the fourt...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/12C12N5/0735C12N5/079C12N5/0793C12N5/0797C12N15/10C12Q1/68
CPCA61K35/12C12N2502/13C12N5/0618C12N5/0619C12N5/0622C12N5/0623C12N15/1034C12N15/1096C12N2501/01C12N2501/105C12N2501/11C12N2501/115C12N2501/119C12N2501/13C12N2501/135C12N2501/155C12N2502/99C12N2503/02C12N2506/02C12N2510/00C12N2510/04C12Q1/6881C12N5/0606Y02A50/30
Inventor CARPENTER, MELISSA K.DENHAM, JERROD J.INOKUMA, MARGARET S.THIES, R. SCOTT
Owner ASTERIAS BIOTHERAPEUTICS INC
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