FIBROBLAST BASED THERAPY FOR TREATMENT OF PARKINSON'S DISEASE

Abstract

In some aspects, disclosed herein are methods and compositions for treatment of Parkinson's disease using fibroblasts or cells derived from fibroblasts. Also disclosed herein are methods and compositions for generating dopaminergic cells from fibroblasts. Dopaminergic cells generated from fibroblasts are described. Methods of the present disclosure include methods for treatment or preventing of Parkinson's disease comprising the use of fibroblasts or dopaminergic cells generated from fibroblasts.

Claims

1. A method for treating or preventing Parkinson's disease in an individual comprising administering an effective amount of a composition comprising fibroblast cells to an individual in need thereof.

2. The method of claim 1, wherein the fibroblast cells are dopaminergic fibroblasts cells.

3. The method of claim 1 or 2, wherein the fibroblast cells are cultured under conditions sufficient to differentiate the fibroblasts into neuronal cells.

4. The method of claim 3, wherein the conditions comprise treatment of the fibroblasts with one or more agents capable of activating tyrosine hydroxylase expression in the fibroblasts.

5. The method of claim 3 or 4, wherein the conditions comprise culturing the fibroblasts with all-trans retinoic acid (RA).

6. The method of any one of claims 2-5, wherein the conditions comprise culturing the fibroblasts with one or more growth factors.

7. The method of claim 6, wherein the growth factor is one or more neurotrophic factors.

8. The method of claim 7, wherein the one or more neurotrophic factors comprise brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), a fibroblast growth factor (FGF), or a combination thereof.

9. The method of any one of claims 1-8, wherein the Parkinson's disease comprises immunologically mediated loss of endogenous dopaminergic cells in the individual.

10. The method of any one of claims 1-9, wherein the Parkinson's disease comprises non-immunologically mediated loss of endogenous dopaminergic cells in the individual.

11. The method of any one of claims 1-10, wherein the dopaminergic cells express tyrosine hydroxylase.

12. The method of claim 11, wherein the dopaminergic cells express the tyrosine hydroxylase transiently.

13. The method of any one of claims 1-12, wherein the dopaminergic cells express NeuN.

14. The method of any one of claims 1-13, wherein the dopaminergic cells do not express CD40.

15. The method of any one of claims 1-14, wherein the dopaminergic cells express Nestin.

16. The method of any one of claims 1-15, wherein the dopaminergic cells are provided to the individual intracranially.

17. The method of claim 16, wherein the dopaminergic cells are provided to the putamen of the individual.

18. The method of any one of claims 1-17, wherein the individual has been diagnosed with Parkinson's disease.

19. The method of any one of claims 1-17, wherein the individual is at risk for developing Parkinson's disease.

20. The method of claim 19, wherein the individual at risk of developing Parkinson's disease includes male individuals, individuals age 60 and older, individuals with a genetic predisposition, individuals who have been exposed to environmental toxins, and/or individuals with a history of head trauma.

21. The method of any one of claims 1-20, wherein the Parkinson's disease comprises corticobasal degeneration, dementia with Lewy bodies, drug-induced parkinsonism, essential tremor, multiple system atrophy, progressive supranuclear palsy, vascular parkinsonism, or a combination thereof.

21. The method of any one of claims 1-21, wherein the fibroblasts are exposed to inflammatory conditions, and wherein the fibroblasts reduce inflammation in the individual.

22. A method for generating dopaminergic cells from fibroblasts comprising subjecting fibroblasts to conditions sufficient to generate dopaminergic cells from the fibroblasts.

23. The method of claim 22, wherein the conditions comprise conditions sufficient to differentiate the fibroblasts into neuronal cells.

24. The method of claim 22 or 23, wherein the conditions comprise treatment of the fibroblasts with one or more agents capable of activating tyrosine hydroxylase expression in the fibroblasts.

25. The method of any one of claims 22-24, wherein the conditions comprise culturing the fibroblasts with all-trans retinoic acid (RA).

26. The method of claim 25, wherein the conditions further comprise culturing the fibroblasts with one or more growth factors.

27. The method of claim 26, wherein the growth factor is one or more neurotrophic factors.

28. The method of claim 27, wherein the one or more neurotrophic factors comprise brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), a fibroblast growth factor (FGF), or a combination thereof.

29. The method of any one of claims 22-28, wherein the dopaminergic cells express tyrosine hydroxylase.

30. The method of claim 29, wherein the dopaminergic cells express the tyrosine hydroxylase transiently.

31. The method of any one of claims 22-30, wherein the dopaminergic cells express NeuN.

32. The method of any one of claims 22-31, wherein the dopaminergic cells do not express CD40.

33. The method of any one of claims 22-32, wherein the dopaminergic cells express Nestin.

34. The method of any one of claims 1-33, wherein the fibroblasts are fibroblasts isolated from placenta, cord blood, peripheral blood, omentum, hair follicle, skin, bone marrow, adipose tissue, or Wharton's Jelly.

35. The method of any one of claims 1-34, wherein the fibroblasts are fibroblasts isolated from peripheral blood of a subject who has been exposed to conditions sufficient to stimulate fibroblasts from the subject to enter the peripheral blood.

36. The method of claim 35, wherein the conditions sufficient to stimulate fibroblasts from the subject to enter the peripheral blood comprise administration of VLA-5 antibodies, G-CSF, M-CSF, GM-CSF, FLT-3L, TNF-α, EGF, FGF-1, FGF-2, FGF-5, VEGF, or a combination thereof.

37. A dopaminergic cell, wherein the dopaminergic cell is derived from a fibroblast that was cultured with all-trans retinoic acid (RA) and one or more neurotrophic factors.

38. The dopaminergic cell of claim 37, wherein the dopaminergic cell expresses tyrosine hydroxylase.

39. The dopaminergic cell of claim 38, wherein the dopaminergic cell expresses the tyrosine hydroxylase transiently.

40. The dopaminergic cell of any one of claims 37-39, wherein the dopaminergic cell expresses NeuN.

41. The dopaminergic cell of any one of claims 37-40, wherein the dopaminergic cell does not express CD40.

42. The dopaminergic cell of any one of claims 37-41, wherein the dopaminergic cell expresses Nestin.

43. The dopaminergic cell of any one of claims 37-42, wherein the one or more neurotrophic factors comprise brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), a fibroblast growth factor (FGF), or a combination thereof.

44. The dopaminergic cell of any one of claims 37-43, wherein the fibroblast was isolated from placenta, cord blood, peripheral blood, omentum, hair follicle, skin, bone marrow, adipose tissue, or Wharton's Jelly.

45. The dopaminergic cell of any one of claims 37-44, wherein the fibroblast was isolated from peripheral blood of a subject who has been exposed to conditions sufficient to stimulate fibroblasts from the subject to enter the peripheral blood.

46. The dopaminergic cell of claim 45, wherein the conditions sufficient to stimulate fibroblasts from the subject to enter the peripheral blood comprise administration of VLA-5 antibodies, G-CSF, M-CSF, GM-CSF, FLT-3L, TNF-α, EGF, FGF-1, FGF-2, FGF-5, VEGF, or a combination thereof.

Description

EXAMPLES

[0090] The following examples are included to demonstrate particular embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the methods of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Example 1

Superior Proliferation of Fibroblasts Compared to Bone Marrow and Adipose-Derived Mesenchymal Stem Cells

[0091] Dermal fibroblasts, bone marrow mesenchymal stem cells (BM-MSCs), and adipose derived mesenchymal stem cells (A-MSCs) where cultured for 7 days in DMEM with 10% fetal calf serum in the presence of 0.03 IU oxytocin per ml. Proliferation was assessed by MTT staining. FIG. 1 shows the results of the proliferation analysis. Significantly greater proliferation was observed for the fibroblast cells compared to the BM-MSC and A-MSC under these conditions. The fibroblast cells doubled once every 18-22 hours, while the BM-MSC doubled every 28-36 hours, and the A-MSC doubled every 25-29 hours.

Example 2

Differentiation of Fibroblasts into Cells Having Neuron-Like Morphology and Phenotype

[0092] Fibroblasts, BM-MSCs and A-MSCs were plated in 6 well polylysine coated plates at a concentration of 50,000 cells per ml. Cells where incubated in induction media containing N2 media supplemented with 0.5 μM all-trans retinoic acid (RA) and 1 ng/ml brain-derived neurotrophic factor (BDNF). Cells where cultured for 7 days in a fully humidified atmosphere with addition of 0.03 IU oxytocin per ml. As shown in FIG. 2, increased NeuN staining as well as neuron-like morphology was observed in cultures from fibroblasts. Furthermore, an additional 3 day culture resulted in further enhancement of neuronal morphology in the fibroblast derived cells but not in the BM-MSCs or A-MSCs.

Example 3

Production of Tyrosine Hydroxylase by Fibroblasts

[0093] Cells were grown as described in Example 1 and 2, and induced to differentiate as described in Example 2, except that cells were cultured for 8 days. Tyrosine hydroxylase was assessed by quantitative real-time polymerase chain reaction (RT-PCR) and expressed as percentage of housekeeping gene GAPDH. As shown in FIG. 3, significant induction of this dopamine-producing enzyme was observed in the fibroblast-derived neuronal cells compared with the BM-MSCs and A-MSCs.

[0094] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.