HK1245659B - Cell therapy for the treatment of neurodegeneration - Google Patents

Description

Cell therapy for treating neurodegeneration

The present application is a divisional application of an invention patent application filed on February 6, 2013, with application number 2013800753669 and the invention name “Cell therapy for treating neurodegeneration”.

Technical Field

The present disclosure describes cellular compositions and methods for treating neurodegeneration.

Background Art

Neurodegeneration is a pathological state that causes the death of nerve cells. Although the causes of neurodegeneration may be varied and not always certain, many neurological disorders have neurodegeneration as a common pathological state. For example, Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (ALS) all cause chronic neurodegeneration, which is characterized in that nerve cells die slowly and gradually over several years, while acute neurodegeneration is characterized in that ischemia (e.g., stroke) or trauma (e.g., traumatic brain injury) causes, or by the sudden onset of nerve cell death caused by demyelination or axonal transection caused by trauma, such as spinal cord injury or multiple sclerosis.

Neurodegeneration can also be triggered by various nerve cell injuries, such as those caused by alcoholism, drug addiction, exposure to neurotoxins, and radiation. Signs of neurodegeneration can even be found in dementia, epilepsy, various psychiatric disorders, and is also part of the normal aging process.

Whatever the underlying cause, growing evidence suggests that once neurodegeneration is initiated, the end result for all of these conditions is the same: the eventual death of nerve cells.

A stroke involves acute neurodegeneration (rapid loss of central nervous system nerve cells with consequent loss of function) caused by a blockage (ischemic stroke) or rupture (hemorrhage) of a blood vessel leading to or within the brain. Because stroke can cause permanent neurological damage, systemic complications, and even death, it often constitutes a medical emergency. In the United States and Europe, stroke is the leading cause of disability among adults and the second leading cause of death worldwide. In the United States, more than one in fifteen people die from stroke, and it ranks third among all causes of death, after heart disease and cancer (American Heart Association, Heart Disease and Stroke Statistics – 2009 Update, Dallas, TX: American Heart Association; 2009; Rosamond et al., Heart Disease and Stroke Statistics – 2007 Update: A Report from the American Heart Association Committee on Statistics and Subcommittee on Stroke Statistics, Circulation, 2007;115:e69-e171). One out of every three strokes is fatal (Walker et al., Risk factors of stroke incidence and mortality, A 12-year follow-up of the Oslo Study, Stroke, 1993 24(10;1484-0). Approximately 6% of all deaths under the age of 65 and 10% of all deaths over the age of 65 are due to stroke (Donnan et al., Stroke, Lancet, 2008;317(9624):1612-23). Thus, statistics demonstrate that for many stroke victims, severe disability is an unfortunate but all too frequent outcome. In fact, stroke is the leading cause of inpatient Medicare claims for long-term adult care. In the United States and other major industrial countries, the total costs associated with stroke treatment and rehabilitation now exceed $45 million per year and undoubtedly continue to contribute to the overall increase in health care costs.

Ischemic stroke is the most common form of stroke, accounting for more than 80% of all strokes. Ischemic stroke is caused by arterial blockage, which is usually due to thrombosis or, less commonly, embolism. The attack is usually sudden and is usually followed by local neurological deficits. About 20% of patients die within a few days, especially if the infarct is large. Another 10% of patients die within a few weeks of their first stroke. Unfortunately, survivors are usually severely disabled. Depending on the area of the brain affected, symptoms include unilateral facial or limb weakness and sensory impairment as well as cognitive and language disorders. The larger the area of the brain affected, the more functions may be impaired. Some functional improvements may begin to appear within a few days, and further recovery is common over a period of several months. However, the extent of recovery is unpredictable and is usually incomplete. According to the American Stroke Association, 15% to 30% of stroke survivors are permanently disabled, and 20% require institutional care within three months of the onset of the disease (Harmsen et al., Long-term risk factors for stroke: twenty-eight years offollow-up of 7457 middle-aged men in Sweden, Stroke, 2006; 37(7): 1663-7).

An ischemic stroke results in a core lesion, where nerve cells die within minutes from lack of oxygen, and a surrounding penumbra. The penumbra receives some blood flow and, therefore, some oxygen, but still less than normal. Cell death in the penumbra proceeds more slowly, typically within hours, and is caused by variable anoxia and the ischemic cascade, along with toxic substances produced by glutamate released within the core lesion. Therefore, current therapeutic interventions primarily focus on mitigating the deleterious effects of the penumbra.

However, therapies for acute ischemic stroke remain limited.

Because brain damage occurs as a result of reduced blood flow to the brain, current treatments aim to remove blocked arteries by either dissolving the clot (thrombolysis) or mechanically removing the clot (thrombectomy). The faster blood flow is restored, the less brain cells die and the greater the chance of avoiding permanent sequelae.

Currently, only two therapies are FDA-approved for stroke in the United States:

Recombinant tissue plasminogen activator (rt-PA; Genentech), a drug that dissolves arterial clots; and

The Merci Retrieval System (Concentric Medical Inc.) and the Penumbra System (Penumbra Inc.), devices that mechanically remove blood clots.

All of the above treatment approaches have major limitations.

To be effective, therapy with thrombolytics must be initiated within 3 to 4.5 hours of symptom onset, meaning only approximately 3% of patients with acute ischemic stroke receive effective rt-PA treatment. Furthermore, thrombolytic therapy carries a substantially increased risk of intracerebral hemorrhage, further limiting its use in some individuals.

For the reasons stated above, there is an unmet and urgent need in the art for safe and effective therapies that mitigate and/or prevent neurodegeneration, particularly neurodegeneration caused by ischemia.

Summary of the Invention

Thus, in one embodiment, the present invention provides a heterogeneous bone marrow (BM) cell subpopulation prepared by the following method, the method comprising:

a) obtaining a bone marrow cell population from untreated bone marrow;

b) seeding a bone marrow cell population at low density on a plastic surface;

c) washing the seeded cell population to remove unattached cells;

d) culturing the attached cell population in serum-containing medium until near confluence;

e) serially passage the cultured cell population for no more than about seven serial passages, wherein at each passage the cultured cells are seeded at a low density; and

f) obtaining a heterogeneous bone marrow cell subpopulation, wherein an effective amount of the heterogeneous bone marrow cell subpopulation is effective to treat neurodegeneration.

In one aspect, an effective amount of a heterogeneous myeloid cell subpopulation is effective to treat neurodegeneration caused by ischemia.

In one aspect, the heterogeneous subpopulation of bone marrow cells is cultured in a serum-containing medium.

In one aspect, the bone marrow cell population is seeded at a density of about 10 ² -10 ⁶ cells/cm ² .

In one aspect, at the time of passaging, the cultured cells are seeded at a density of about 750 cells/ ^cm2 or less.

Untreated bone marrow can be obtained from a subject that has not been pretreated with any agents that regulate cell division, such as antineoplastic drugs used in chemotherapy, including antimetabolites such as 5-fluorouracil.

In one aspect, the heterogeneous myeloid cell subpopulation cannot be separated by density separation methods such as Ficoll ^™ or Percoll ^™ gradients or by ACK (ammonium-chloride-potassium) lysis buffer.

In a further aspect of the present invention, the heterogeneous myeloid cell subpopulation is further tested in an experimental model of the neurodegeneration caused by ischemia, wherein only those cell populations that show the ability to treat neurodegeneration are selected. The experimental model of the neurodegeneration caused by ischemia can be an oxygen/glucose deprivation (OGD) cell culture model or a stroke animal model. In one aspect, the experimental model can be an oxygen/glucose deprivation (OGD) cell culture model and a subsequent stroke animal model.

In another aspect, following injection into the bloodstream, cells in the heterogeneous subpopulation migrate to sites of neurodegeneration.

In one aspect, the neurodegeneration caused by ischemia is ischemic stroke.

In another embodiment, the present invention also provides a method for treating neurodegeneration caused by ischemia, comprising injecting the above-mentioned heterogeneous bone marrow cell subpopulation into the bloodstream of a mammal suffering from neurodegeneration, wherein the injection of the heterogeneous bone marrow cell subpopulation reduces the neurological function deficit caused by neurodegeneration.

When the neurodegeneration is caused by an ischemic stroke, the method may further include an additional therapy for increasing blood flow through the blocked blood vessel, wherein the combination of injecting the heterogeneous bone marrow cell subset and the additional therapy results in a greater reduction in neurological deficits than following the additional therapy alone or injecting the heterogeneous bone marrow cell subset without the additional therapy.

In one aspect, an additional treatment to increase blood flow through the blocked blood vessel is administered concurrently with the injection of the heterogeneous bone marrow cell subpopulation.

In another aspect, an additional treatment to increase blood flow through the blocked blood vessel is administered prior to injection of the heterogeneous bone marrow cell subpopulation.

When neurodegeneration is caused by an ischemic stroke, a blood vessel may be blocked by a blood clot, which may result from the rupture of an atherosclerotic plaque.

When the neurodegeneration is caused by an ischemic stroke, additional treatment to increase blood flow through the blocked blood vessel may include administering a thrombolytic agent. The thrombolytic agent may be at least one of streptokinase, urokinase, and recombinant tissue plasminogen activator. Furthermore, the recombinant tissue plasminogen activator may be alteplase, reteplase, desmoteplase, or tenecteplase.

In another aspect, when the neurodegeneration is caused by an ischemic stroke, additional treatment to increase blood flow through the blocked blood vessel may include mechanically removing the blood clot from the blocked blood vessel.

In a further aspect, a filter can be used to capture blood clots.

In another aspect, when the neurodegeneration is caused by an ischemic stroke, additional treatment to increase blood flow through the blocked blood vessel may include performing angioplasty and/or stenting.

The heterogeneous myeloid cell subset can be injected intravenously, intraarterially (eg, into the carotid artery), or intracerebrally.

In another embodiment, the present invention also provides a method for treating ischemia-induced neurodegeneration, comprising injecting a heterogeneous bone marrow cell subpopulation into the bloodstream of a mammal suffering from ischemia-induced neurodegeneration, wherein the injection of the heterogeneous bone marrow cell subpopulation reduces the volume of the infarct caused by the ischemia.

When the neurodegeneration is caused by an ischemic stroke, the method may further include an additional therapy for increasing blood flow through the blocked blood vessel, wherein the combination of injecting the heterogeneous bone marrow cell subset and the additional therapy results in a greater reduction in infarct volume than following the additional therapy alone or injecting the heterogeneous bone marrow cell subset without the additional therapy.

In one aspect, when the neurodegeneration is caused by ischemic stroke, the additional treatment for increasing blood flow through the blocked blood vessel comprises administering a thrombolytic agent. The thrombolytic agent can be at least one of streptokinase, urokinase, and recombinant tissue plasminogen activator. Furthermore, the recombinant tissue plasminogen activator can be alteplase, reteplase, desmoteplase, or tenecteplase.

In another aspect, when the neurodegeneration is caused by an ischemic stroke, additional treatment to increase blood flow through the blocked blood vessel may include performing angioplasty and/or stenting procedures.

In another aspect, when neurodegeneration results from an ischemic stroke, additional treatment to increase blood flow through the blocked vessel may include mechanical removal of the blood clot near the infarct site.

According to a second approach, a heterogeneous subset of bone marrow cells can be injected intravenously, intraarterially (eg, into the carotid artery), or intracerebrally.

When neurodegeneration is caused by ischemic stroke, the present invention further provides a kit comprising a thrombolytic agent and the aforementioned heterogeneous bone marrow cell subset. The thrombolytic agent may be at least one of streptokinase, urokinase, and recombinant tissue plasminogen activator. Furthermore, the recombinant tissue plasminogen activator may be alteplase, reteplase, or tenecteplase.

When neurodegeneration is caused by ischemic stroke, the present invention may also provide a kit comprising the heterogeneous bone marrow cell subpopulation described above and a tool for mechanically removing blood clots.

Furthermore, when neurodegeneration is caused by ischemic stroke, the present invention can provide a composition for treating ischemic neurodegeneration, comprising a thrombolytic agent, the aforementioned heterogeneous bone marrow cell subset, and a carrier for injecting the composition. The thrombolytic agent can be at least one of streptokinase, urokinase, and recombinant tissue plasminogen activator. Furthermore, the recombinant tissue plasminogen activator can be alteplase, reteplase, or tenecteplase.

The present invention may also provide a method for preparing a heterogeneous bone marrow cell subpopulation for treating neurodegeneration, comprising:

a) obtaining a heterogeneous population of bone marrow cells from untreated bone marrow;

b) seeding a heterogeneous population of bone marrow cells at low density on a plastic surface;

c) washing the seeded cell population to remove unattached cells;

d) culturing the attached cells from the washed cell population in serum-containing medium to near confluence;

e) serially passage the cultured cells for no more than about seven serial passages,

Here, at each passage, cultured cells are seeded at low density, thereby obtaining heterogeneous bone marrow cell subsets.

In one aspect, such untreated bone marrow can be obtained from a subject that has not been pretreated with any agents that regulate cell division, such as anti-tumor drugs used in chemotherapy, including antimetabolites, such as 5-fluorouracil.

In one aspect, the heterogeneous myeloid cell subpopulation cannot be separated by density separation methods such as Ficoll ^™ or Percoll ^™ gradients or by ACK (ammonium-chloride-potassium) lysis buffer.

In a further aspect of the present invention, the heterogeneous bone marrow cell subpopulation can be further tested in the neurodegeneration experimental model caused by ischemia, wherein only those cell populations that show the ability to treat the neurodegeneration caused by ischemia are selected. The neurodegeneration experimental model caused by ischemia can be an oxygen/glucose deprivation (OGD) cell culture model and a stroke animal model. In one aspect, the experimental model can be an oxygen/glucose deprivation (OGD) cell culture model and a subsequent stroke animal model. In one aspect, both models can be used for testing heterogeneous bone marrow cell subpopulations.

In another aspect, the present invention may also provide a method for optimizing an experimental protocol for isolating a heterogeneous cell population for treating ischemia-induced neurodegeneration, comprising the following steps:

Isolate cell populations according to the experimental protocol;

wherein the cell population treats neurodegeneration caused by ischemia; and

Optimal parameters for each step of the protocol were determined by testing their effect on the efficacy of isolated cell populations in treating ischemia-induced neurodegeneration in an experimental model of ischemia.

In one aspect, parameters may include cell density at seeding, cell passage number, culture medium composition, or cell isolation method.

In one aspect, the ischemic experimental model can be an oxygen/glucose deprivation (OGD) cell culture model or a stroke animal model, such as a middle cerebral artery occlusion (MCAO) animal model. Similarly, both models can be used. In one aspect, the experimental model can be an oxygen/glucose deprivation (OGD) cell culture model and a subsequent stroke animal model.

The above aspects have many advantages, including the ability of a heterogeneous bone marrow cell subset to treat neurodegeneration caused by ischemic stroke. The cell population significantly reduces infarct size and improves neurological function.

Combined with thrombolytics and mechanical clot removal methods, this cell therapy has the potential to have a significant clinical impact on patient survival, functional function, and quality of life after stroke.

The present invention also relates to the following items:

1. A heterogeneous bone marrow cell subset prepared by the following method, the method comprising:

a) obtaining a bone marrow cell population from untreated bone marrow;

b) seeding the bone marrow cell population at a low density on a plastic surface;

c) washing the seeded cell population to remove unattached cells;

d) culturing the adherent cell population in serum-containing medium until near confluence; serially passage the cultured cell population for no more than about seven serial passages, wherein, at each passage, the cultured cells are seeded at a low density; and

e) obtaining the heterogeneous bone marrow cell subpopulation, wherein an effective amount of the heterogeneous bone marrow cell subpopulation is effective to treat neurodegeneration.

2. The heterogeneous bone marrow cell subpopulation according to item 1, wherein the neurodegeneration is caused by ischemia.

3. The heterogeneous bone marrow cell subpopulation according to item 2, which is further tested in an experimental model of neurodegeneration, wherein those cell populations are selected that show the ability to treat neurodegeneration.

4. The heterogeneous bone marrow cell subpopulation according to item 3, wherein the neurodegeneration experimental model is an oxygen/glucose deprivation (OGD) cell culture model.

5. The heterogeneous bone marrow cell subpopulation according to item 3, wherein the neurodegeneration experimental model is a stroke animal model.

6. The heterogeneous bone marrow cell subpopulation according to item 1, wherein the bone marrow cell population is seeded at a density of about 10 ² -10 ⁶ cells/cm ² .

7. The heterogeneous bone marrow cell subpopulation according to item 1, wherein the obtained bone marrow cell subpopulation is cultured in a serum-containing medium.

8. The heterogeneous bone marrow cell subpopulation according to item 1, wherein the cultured cells are seeded at a density of less than about 750 cells/ ^cm2 at each passage.

9. The heterogeneous bone marrow cell subpopulation according to item 1, wherein upon injection into the bloodstream, cells in the subpopulation migrate to sites of neurodegeneration.

10. The heterogeneous bone marrow cell subpopulation according to claim 1, wherein the untreated bone marrow is obtained from a subject who has not been pretreated with a chemotherapeutic agent.

11. The heterogeneous bone marrow cell subpopulation according to item 10, wherein the chemotherapeutic agent is 5-fluorouracil.

12. The heterogeneous bone marrow cell subpopulation according to item 1, wherein the bone marrow cell population cannot be separated by density separation or by ACK lysis buffer.

13. The heterogeneous bone marrow cell subpopulation according to item 12, wherein the density separation method requires a Ficoll ^™ or Percoll ^™ gradient.

14. A method for treating neurodegeneration, comprising:

injecting the heterogeneous bone marrow cell subpopulation according to item 1 into the bloodstream of a mammal suffering from neurodegeneration,

wherein said injection of said bone marrow cell subpopulation reduces neurological deficits caused by said neurodegeneration.

15. The method for treating neurodegeneration according to item 14, wherein the neurodegeneration is caused by ischemic stroke.

16. The method for treating neurodegeneration according to item 15, further comprising an additional treatment for increasing blood flow through the blocked blood vessel,

wherein the injection of the bone marrow cell subset in combination with the additional therapy results in a greater reduction in neurological deficit than following the additional therapy alone or following the injection of the bone marrow cell subset without the additional therapy.

17. The method for treating neurodegeneration according to item 16, wherein the additional treatment for increasing blood flow through the blocked blood vessel is performed simultaneously with the injection of the bone marrow cell subpopulation.

18. The method for treating neurodegeneration according to item 16, wherein the additional treatment for increasing blood flow through the blocked blood vessel is performed before injecting the bone marrow cell subpopulation.

19. The method for treating neurodegeneration according to item 16, wherein the blood vessel is blocked by a blood clot.

20. The method for treating neurodegeneration according to item 20, wherein the blood clot is produced by rupture of an atherosclerotic plaque.

21. The method for treating neurodegeneration according to item 16, wherein the additional treatment for increasing blood flow through the blocked blood vessel comprises administering a thrombolytic agent.

22. The method for treating neurodegeneration according to item 21, wherein the thrombolytic agent is at least one selected from streptokinase, urokinase and recombinant tissue plasminogen activator.

23. The method for treating neurodegeneration according to item 22, wherein the recombinant tissue plasminogen activator is alteplase, reteplase, desmoteplase or tenecteplase.

24. The method for treating neurodegeneration according to item 16, wherein the additional treatment for increasing blood flow through the blocked blood vessel comprises mechanically removing the blood clot from the blocked blood vessel.

25. The method for treating neurodegeneration according to item 16, wherein the additional treatment for increasing blood flow through the blocked blood vessel further comprises using a filter to capture the blood clot.

26. The method for treating neurodegeneration according to item 16, wherein the additional treatment for increasing blood flow through the blocked blood vessel comprises performing angioplasty and/or vascular stenting.

27. The method for treating neurodegeneration according to item 14, wherein the bone marrow cell subpopulation is injected intravenously or intraarterially.

28. The method for treating neurodegeneration according to item 14, wherein the bone marrow cell subpopulation is injected into the carotid artery.

29. The method for treating neurodegeneration according to item 14, wherein the bone marrow cell subpopulation is injected into the brain.

30. A method for treating neurodegeneration caused by ischemic stroke, comprising:

injecting the heterogeneous bone marrow cell subpopulation according to item 1 into the bloodstream of a mammal suffering from ischemia-induced neurodegeneration,

wherein said injection of said bone marrow cell subpopulation reduces infarct volume caused by said neurodegeneration.

31. The method for treating neurodegeneration according to item 30, further comprising an additional treatment for increasing blood flow through the blocked blood vessel,

wherein the combination of injection of the bone marrow cell subset and the additional therapy results in a greater reduction in infarct volume than the additional therapy alone or injection of the bone marrow cell subset without the additional therapy.

32. The method for treating neurodegeneration according to item 31, wherein the additional treatment for increasing blood flow through the blocked blood vessel comprises administering a thrombolytic agent.

33. The method for treating neurodegeneration according to item 32, wherein the thrombolytic agent is at least one selected from streptokinase, urokinase and recombinant plasminogen activator.

34. The method for treating neurodegeneration according to item 33, wherein the recombinant plasminogen activator is alteplase, reteplase, desmoteplase or tenecteplase.

35. The method for treating neurodegeneration according to item 31, wherein the additional treatment for increasing blood flow through the blocked blood vessel comprises performing angioplasty and/or stenting.

36. The method for treating neurodegeneration according to item 31, wherein the additional treatment for increasing blood flow through the blocked blood vessel comprises mechanically removing the blood clot near the infarct site.

37. The method for treating neurodegeneration according to item 30, wherein the bone marrow cell subpopulation is injected intravenously or intraarterially.

38. The method for treating neurodegeneration according to item 30, wherein the bone marrow cell subpopulation is injected into the carotid artery.

39. The method for treating neurodegeneration according to item 30, wherein the bone marrow cell subpopulation is injected into the brain.

40. A kit comprising a thrombolytic agent and the heterogeneous bone marrow cell subpopulation according to item 1.

41. The kit according to item 40, wherein the thrombolytic agent is at least one selected from streptokinase, urokinase and recombinant tissue plasminogen activator.

42. The kit according to item 41, wherein the recombinant tissue plasminogen activator is alteplase, reteplase or tenecteplase.

43. A kit comprising the heterogeneous bone marrow cell subpopulation according to item 1 and a tool for mechanically removing blood clots.

44. A composition for treating neurodegeneration caused by ischemic stroke, comprising a thrombolytic agent, the bone marrow cell subpopulation according to item 1, and a carrier for injecting the composition.

45. The composition according to item 44, wherein the thrombolytic agent is at least one selected from streptokinase, urokinase and recombinant tissue plasminogen activator.

46. The composition according to item 45, wherein the recombinant tissue plasminogen activator is alteplase, reteplase or tenecteplase.

47. A method for preparing a heterogeneous bone marrow cell subpopulation for treating neurodegeneration, comprising:

a) obtaining a bone marrow cell population from untreated bone marrow;

b) seeding the bone marrow cell population at a low density on a plastic surface;

c) washing the seeded cell population to remove unattached cells;

d) culturing the attached cells from the washed cell population in serum-containing medium to near confluence;

e) serially passage the cultured cells for no more than about seven serial passages, wherein at each passage, the cultured cells are seeded at a low density; and

f) obtaining the heterogeneous bone marrow cell subpopulation, wherein the heterogeneous bone marrow cell subpopulation is used to treat neurodegeneration.

48. The method of claim 47, wherein the untreated bone marrow is obtained from a subject that has not been pretreated with a chemotherapeutic agent.

49. The method of claim 48, wherein the chemotherapeutic agent is 5-fluorouracil.

50. The method according to item 48, wherein the bone marrow cell population cannot be separated by density separation or by ACK lysis buffer.

51. The method of item 50, wherein the density separation method requires a Ficoll ^™ or Percoll ^™ gradient.

52. The method of claim 47, wherein the neurodegeneration is caused by ischemia.

53. The method of item 47, further comprising testing the heterogeneous myeloid cell subpopulations in an experimental model of neurodegeneration, wherein only those populations that demonstrate the ability to treat neurodegeneration are selected.

54. The method according to item 53, wherein the experimental model is an ischemia experimental model.

55. The method according to item 54, wherein the ischemia experimental model is an oxygen/glucose deprivation (OGD) cell culture model.

56. The method according to item 54, wherein the experimental model is an animal model of stroke.

57. A method for optimizing an experimental protocol for isolating a cell population for treating neurodegeneration, comprising:

Isolate cell populations according to the experimental protocol;

wherein the cell population treats neurodegeneration; and

The optimal parameters for each step of the protocol are determined by testing the effect of each parameter on the therapeutic effect of the isolated cell population on neurodegeneration in an experimental animal model of neurodegeneration.

58. The method of claim 57, wherein the neurodegeneration is caused by ischemic stroke.

59. The method according to item 57, wherein the parameters include cell density at the time of inoculation, cell passage number, culture medium composition or cell separation method.

60. The method according to item 57, wherein the neurodegeneration experimental model is an ischemia experimental model.

61. The method according to item 60, wherein the ischemia experimental animal model is an oxygen/glucose deprivation (OGD) cell culture model.

62. The method according to item 60, wherein the ischemia experimental animal model is a stroke animal model.

63. The method according to item 60, wherein the ischemia experimental model is a middle cerebral artery occlusion (MCAO) animal model.

BRIEF DESCRIPTION OF THE DRAWINGS

Those skilled in the art will appreciate that the following drawings are for illustration purposes only and are not intended to limit the scope of the teachings in any way.

FIG1 depicts the results of screening for candidate cell subsets derived from bone marrow using an in vitro OGD model ( FIG1A , FIG1B , and FIG1E ) and an in vivo MCAO rat model ( FIG1C , FIG1D , and FIG1F ).

Figures 1A and 1B depict host cell survival and cytokine release (bFGF and IL-6), respectively, in an in vitro OGD model in response to the presence or absence of seven different candidate cell subsets derived from bone marrow.

Figures 1C and 1D compare host cell survival, infarct volume, and neurological function in an in vivo MCAO rat model in response to intravenous (IV) or intra-arterial (ICA) injection of NCS-01 bone marrow cell subsets or saline.

FIG. 1E depicts host cell survival and cytokine release (bFGF and IL-6) in an in vitro OGD model in response to the presence or absence of NCS-01 myeloid cell subsets.

FIG. 1F shows changes in infarct volume and neurological function in an in vivo MCAO rat model in response to injection of 3×10 ⁵ , 10 ⁶ , and 10 ⁷ NCS-01 bone marrow cells.

FIG2 shows changes in infarct volume (A) and neurological function (B, measured by the Modified Bederson Scale (0 = normal to 3 = most severe)) in ^a rat model of transient (60 minutes) or permanent MCAO in response to ICA injection of 1 ml of 7.5×10 6 NCS-01 cells or saline solution.

FIG3 shows a schematic diagram describing the protocol used in the in vitro oxygen glucose deprivation (OGD) assay.

FIG4 shows the relative levels of secreted bFGF and IL-6 cytokines in the culture medium of cells analyzed by OGD after addition of saline, Li cells, or NCS-01 cells.

5A and 5B depict host cell survival, infarct volume, and neurological function in an in vivo MCAO rat model in response to injection of saline, Li cells, and NCS-01 cells.

Detailed Description of the Invention

Unless otherwise indicated, the practice of the present invention employs conventional molecular biology techniques within the skill of the art. Such techniques are well known to those skilled in the art and are fully explained in the literature. See, for example, Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y. (1989).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. This specification also provides term definitions to help interpret the disclosure and claims of this application. In the event that a definition is inconsistent with a definition elsewhere, the definition described in this application will prevail.

Cells derived from bone marrow (BM) include a heterogeneous mixture of many different types of cells. The bone marrow is composed of two major cell systems belonging to two different lineages - hematopoietic tissue and an associated support matrix. Therefore, it is known that at least two different stem cells coexist in the bone marrow, namely hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) (Bianco, M. Riminucci, S. Gronthos and P. G. Robey, "Bone marrow stromal stem cells: nature, biology, and potential applications," Stem Cells, Vol. 19, No. 3, pp. 180-192, 2001).

MSCs can be broadly defined by both cell surface markers and by their ability to adhere to tissue/cell culture plastics. Therefore, MSC populations are necessarily heterogeneous, i.e., not clonal cell populations. Even if MSC subpopulations share one or more common cell surface markers, these subpopulations may differ significantly in their biological activities, depending on the production methods used to isolate them. The present disclosure describes a two-step screening protocol that can be used to identify heterogeneous myeloid cell populations that are effective in treating neurodegeneration, including acute onset neurodegeneration, caused by ischemia.

In the first step, bone marrow subpopulations were screened for their ability to mitigate the effects of oxygen glucose deprivation (OGD) in co-cultures containing candidate bone marrow subpopulations and neural cells. The activity of candidate bone marrow subpopulations in mitigating neurodegeneration was assessed by measuring the secretion of trophic factors (bFGF and IL-6) in the culture medium and by determining host cell survival in an OGD assay.

In a second step, the bone marrow-derived cell populations with the strongest activity in the OGD assay were further screened by testing them in an in vivo MCAO rat model of ischemia-induced neurodegeneration. This screening procedure helped identify a heterogeneous bone marrow subpopulation, termed NCS01, that significantly mitigated ischemia-induced neurodegeneration, as defined herein.

Unmanipulated bone marrow cells are readily available to those skilled in the art.

As used herein, the term "unprocessed bone marrow" refers to human bone marrow aspirate that has not undergone any additional processing such as density separation or cell sorting.

In other embodiments, "naive bone marrow" is obtained from a subject that has not been pretreated with any agent that interferes with normal cell growth and division, including, for example, chemotherapeutic agents, such as antimitotic or antimetabolite agents.

Examples of such agents can be found in Cancer Principles and Practice of Oncology, V. T. Devita and S. Hellman (eds.), 6th ed. (February 15, 2001), Lippincott Williams & Wilkins Publishers.

As used herein, an "antimetabolite" agent refers to a compound that inhibits or interrupts DNA synthesis, leading to cell death. Examples of antimetabolites include, but are not limited to, 6-mercaptopurine; cytarabine; fludarabine; floxuridine; 5-fluorouracil; capecitabine; raltitrexed; methotrexate; cladribine; gemcitabine; gemcitabine hydrochloride; thioguanine; hydroxyurea; DNA demethylating agents such as 5-azacytidine and decitabine; edatrexate; and folic acid antagonists such as, but not limited to, pemetrexed.

As used herein, the term "density separation" refers to a well-known laboratory procedure for separating bone marrow cells based on cell density using a Ficoll-Paque ^™ or Percoll ^™ gradient. For example, Ficoll-Paque ^™ is placed at the bottom of a conical tube, and then a layer of untreated bone marrow is slowly overlaid on top of the Ficoll-Paque ^™ . After the cells are centrifuged to pass through the Ficoll gradient, they are separated into several layers based on density, from top to bottom: plasma and other components; a mononuclear cell layer called a buffy coat, which contains peripheral blood mononuclear cells (PBMCs) and mononuclear cells (MNCs), and red blood cells and granulocytes in the pellet. This separation procedure separates red blood cells from PBMCs. Ethylenediaminetetraacetic acid (EDTA) and heparin are often used in conjunction with Ficoll-Paque ^™ to prevent clotting.

As used herein, the term "ACK lysis" refers to an ammonium chloride potassium lysis buffer (ACK lysis buffer) used to lyse red blood cells in EDTA-anticoagulated whole blood.

As used herein, the term "seeding a bone marrow cell population at low density" refers to the concentration of bone marrow cells added at the start of cell culture. In one aspect, the bone marrow cells are seeded at a density of about 10 ² or about 10 ³ or about 10 ⁴ or about 10 ⁵ or about 10 ⁶ cells/cm ^2. In a preferred aspect, the bone marrow cells are seeded at a density of about 10 ⁵ to about 10 ⁶ cells/cm ² .

As used herein, "neurodegeneration" refers to any pathological condition that results in a gradual loss of structure or function of neural cells, including neuronal cell death. Thus, neurodegeneration is a pathological condition caused by a neural disorder.

In one embodiment, the phrase "neural cell" includes nerve cells (i.e., neurons, such as monopolar, bipolar, or multipolar neurons) and their precursors as well as glial cells (e.g., macroglial cells, such as oligodendrocytes, Schwann cells, and astrocytes or microglia) and their precursors.

In one embodiment, an "effective amount" refers to the optimal number of cells required to cause a clinically significant improvement in symptoms and/or pathological conditions associated with neurodegeneration (including slowing, halting, or reversing neurodegeneration, reducing neurological deficits, or improving neurological responses). In some embodiments, an effective amount of the NCS-01 cell population refers to the optimal number of cells required to reduce the infarct volume caused by the sudden onset of acute neurodegeneration after stroke. Those skilled in the art can determine the appropriate effective amount of the NCS-01 cell population for a particular organism using routine experimentation.

As used herein, "treating neurodegeneration" refers to treating neurodegeneration by the NCS-01 cell population, resulting in clinically significant improvement in symptoms and/or pathological conditions associated with neurodegeneration, including slowing, stopping or reversing neurodegeneration, reducing neurological deficits or improving neurological responses.

As used herein, "thrombolytic agent" refers to a drug used medically to dissolve blood clots in a procedure called thrombolysis. Non-limiting examples of thrombolytic drugs include tissue plasminogen activator tPA, alteplase (Activase), reteplase (Retavase), tenecteplase (TNKase), anistreplase (Eminase), streptokinase (Kabikinase, Streptase) and urokinase (Abbokinase).

Described below are procedures for isolating and characterizing heterogeneous myeloid cell subpopulations from unprocessed whole bone marrow that are optimal for treating neurodegeneration, including that caused by ischemia.

Isolation of candidate bone marrow cell populations for effective treatment of ischemia-induced neurodegeneration

Unmanipulated whole bone marrow is obtained from mammals that have not been pretreated with antimitotic or antimetabolite agents, such as 5-fluorouracil (5-FU).

The untreated bone marrow is then directly plated on tissue/cell culture plastic and serially passaged in serum-containing culture medium to amplify. At each passage, cells are seeded at an extremely low cell density, i.e., approximately 750 cells/ ^cm or less, and cultured to near confluence before additional passage. Unattached cells are removed by washing. Since the bone marrow is not processed by density separation, the initial whole bone marrow cell population includes both hematopoietic and non-hematopoietic cells, as well as nucleated bone marrow cells and non-nucleated bone marrow cells.

Then, preferably at passage 3 and passage 5, respectively, a master cell bank (MCB) and a working cell bank (WCB) are established and stored refrigerated. When needed, WCB cells are seeded at very low densities, e.g., about 750 cells/ ^cm2 or less, and expanded in serum-containing medium before being harvested and stored refrigerated using standard procedures.

Two-step selection protocol for evaluating the ability of bone marrow cell populations to mitigate neurodegeneration

The "candidate" bone marrow cell populations can then be screened using a two-step procedure to select the optimal bone marrow cell population for treating neurodegeneration.

In the first step, candidate bone marrow cell populations are tested in an in vitro oxygen-glucose deprivation assay, in which candidate bone marrow cell populations are co-cultured with neural cells under experimental conditions that mimic neurodegeneration. Cell populations that demonstrate in vitro reduction of neurodegeneration are then screened for their ability to treat neurodegeneration in vivo.

In a second step, the selected candidate bone marrow cell populations are evaluated in a rat MCAO model of ischemia-induced neurodegeneration. The candidate heterogeneous bone marrow cell populations that exhibit the highest activity in reducing neurodegeneration in vivo are then selected.

The heterogeneous bone marrow cell population selected by this two-step screening procedure is referred to as the NCS-01 cell population or NCS-01.

Identification of optimal cell culture conditions for isolating bone marrow cell populations with neurodegeneration-alleviating activity

This two-step screening procedure can also be used to identify optimal experimental conditions, such as cell density at seeding, cell passage number, culture medium composition, or cell isolation method for culturing bone marrow cell populations capable of treating neurodegeneration.

Thus, using this two-step procedure, the optimal reseeding cell concentration at each passage is about 750 cells/ ^cm2 or less. The optimal culture medium is serum-containing culture medium. Starting from the initial plating of untreated whole bone marrow, the NCS-01 cell population can be passaged no more than about 7 times, or about 6 times, or about 5 times, or about 4 times, or about 3 times, or about 2 times. Extended passages, i.e., more than 7 passages, from the initial plating of untreated whole bone marrow, reduce or eliminate the ability of NCS-01 to treat neurodegeneration in OGD in vitro assays and in the MCAO rat model.

OGD analysis and the MCAO rat model of neurodegeneration are now described in detail below.

In vitro screening of bone marrow cell populations capable of treating neurodegeneration

Candidate myeloid cell populations were first screened for their ability to prevent neurodegeneration in neuron-astrocyte co-cultures following oxygen-glucose deprivation (OGD) culture conditions that mimic ischemia-induced neurodegeneration.

First, primary mixed cultures of rat or human neurons and astrocytes are exposed to oxygen glucose deprivation (OGD) culture conditions (e.g., 8% oxygen, glucose-free culture medium) for about 0.5 to 3 hours to induce neurodegeneration. The oxygen glucose deprivation of the cell culture is then interrupted, and the OGD-induced neural cells are cultured under physiological conditions for 2 hours, and then co-cultured for another 3 hours in the presence of a candidate bone marrow cell population. Neurodegeneration is then assessed at 0 and 5 hours after OGD by measuring the host cell viability in the presence or absence of a candidate bone marrow cell population. Host cell (neuron and astrocyte) viability can be assessed using, for example, trypan blue staining and/or MTT (bromide-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) analysis.

The cell viability in the presence of the candidate bone marrow cell population is increased by about 5%, or about 10%, or about 15%, or about 20%, or about 25% or more compared to the cell viability of the control (no addition of the candidate bone marrow cell population), indicating that the candidate bone marrow cell population can protect and rescue neuron/astrocyte co-culture from neurodegeneration caused by oxygen and glucose deprivation.

Additionally, commercially available ELISA kits can be used to analyze the induced secretion of trophic factors such as bFGF and/or IL-6 from culture media of neuronal/astrocyte cultures subjected to OGD conditions in the presence or absence of candidate myeloid cell populations.

In certain embodiments, candidate bone marrow cell populations are selected based on their ability to induce an increase in the amount of secreted bFGF and/or IL-6 in the culture medium of a neuron-astrocyte co-culture in response to oxygen-glucose deprivation, but not in the absence of oxygen-glucose deprivation.

In other embodiments, the candidate bone marrow cell population is selected based on its ability to induce at least a two-fold or greater increase in the amount of bFGF and/or IL-6 secreted into the culture medium of a neuron-astrocyte co-culture in response to oxygen-glucose deprivation but not in the absence of oxygen-glucose deprivation.

Candidate bone marrow cell populations that reduced the incidence of OGD-induced cell death and induced increased secretion of trophic factors such as bFGF and/or IL-6 were then selected for screening in an in vivo MCAO rat model (see below).

For example, if a candidate bone marrow cell population reduces the incidence of OGD-induced cell death by more than about 25% and increases the amount of secreted trophic factors by at least 10% or more, the candidate bone marrow cell population can be selected for further screening.

In vivo screening of candidate bone marrow cell populations for the treatment of neurodegeneration

The candidate bone marrow cell population selected in the above OGD analysis screening step is tested for its ability to treat neurodegeneration in vivo. For example, the candidate bone marrow cell population can be tested for its ability to treat neurodegeneration in experimental animal models of neurodegeneration, including transgenic models of neurodegenerative diseases (see, e.g., Harvey et al., Transgenic animal models of neurodegeneration based on human genetic studies, J Neural Transm. (2011) 118(1):27-45; Trancikova et al., Genetic mouse models of neurodegenerative diseases. Prog Mol Biol Transl Sci. (2011), 100:419-82; Chan et al., Generation of transgenic monkeys with human inherited genetic disease Methods (2009) 49(1):78-84; Rockenstein et al., Transgenic animal models of neurodegenerative diseases and their application to treatment development Adv Drug Deliv Rev. (2007) 59(11):1093-102).

In one embodiment, the experimental animal model of neurodegeneration can be a stroke/cerebral ischemia animal model (reviewed by Graham et al., Comp Med. 2004 54(5):486-96), such as the MCAO rat model, in which constriction of a surgically implanted ligature around a cerebral artery simulates the effects of ischemic stroke by restricting blood flow to the brain and causing ischemia and subsequent neurodegeneration.

In the transient MCAO model, the candidate bone marrow cell population selected in the OGD analysis is administered by constant rate infusion into the bloodstream of transient MCAO rats. Those skilled in the art can determine a suitable dose ranging from, for example, 7.5 x 10 ⁴ to 3.75 x 10 ⁷ cells. The cells can be injected, for example, into the jugular vein (IV) or carotid artery (ICA). Controls consist of administering an equal volume of refrigerated culture medium or saline solution. The cells in the candidate bone marrow population then migrate to the site of infarction caused by transient MCAO.

Then, at different times after infarction, neurological function in the presence or absence of the bone marrow cell populations selected by OGD was assessed using the modified Bederson Neurologic Test. Subsequently, the rats were sacrificed and brain tissue sections from treated and untreated MCAO rats were stained with hematoxylin and eosin (H&E) or Nissl staining to measure infarct volume and host cell survival. Bone marrow cell populations were then selected based on their ability to improve neurological function, increase host cell survival, and reduce infarct volume compared to control animals up to 28 days after MCAO.

Combination therapy for treating neurodegeneration caused by ischemic stroke

Stopping blood flow through the middle cerebral artery for an extended period of time simulates permanent arterial occlusion by a blood clot. Temporary occlusion, which stops blood flow through a cerebral artery for a limited period of time before allowing reperfusion to resume, is intended to simulate therapies such as thrombolysis or mechanical clot removal that restore blood flow to the stroke penumbra immediately following arterial occlusion caused by an ischemic stroke.

In many aspects, administration of the NCS-01 cell population to rats with transient MCAO mimics reperfusion of the occluded artery caused by thrombolysis or mechanical clot removal, including angioplasty or surgical stenting.

When neurodegeneration is caused by ischemic stroke, the NCS-01 cell population can be combined with thrombolytic therapy to treat neurodegeneration caused by ischemia. Descriptions of thrombolytic agents and their administration can be found, for example, in US Patent Nos. 5,945,432 and 6,821,985.

Thrombolytic agents injected after an ischemic event can be administered before, simultaneously with, or after the injection of the NCS-01 cell population.

Non-limiting examples of mechanical procedures for improving blood flow to the stroke penumbra that can be combined with injection of the disclosed NCS-01 cell compositions include angioplasty or stent implantation, according to procedures well known in the art.

The present invention will be described in further detail with reference to the following examples.

Example

The examples set forth methods for isolating, selecting, and using bone marrow cell subsets for treating neurodegeneration according to the present invention. It should be understood that the method steps described in these examples are not intended to be limiting. Further objects and advantages of the present invention, in addition to those described above, will be apparent from the examples, which are not intended to limit the scope of the invention.

Example 1: Isolation of NCS-01 cell population

1) Isolation of candidate bone marrow cell populations

Isolate a heterogeneous population of bone marrow cells using the following production methods:

Untreated human bone marrow was harvested from pre-screened healthy donors aged 50 years or younger provided by qualified commercial suppliers. Bone marrow was harvested from donors who had not been pretreated with any anti-mitotic agents such as 5-fluorouracil.

• The treated or untreated bone marrow is then plated at low density (10 ² -10 ⁶ cells/cm ² ) onto a tissue/cell culture plastic surface and cultured in the presence of serum-containing medium;

After allowing the cells to attach to the plastic for several days, removing unattached cells by washing; and

- culturing the adherent cell population to near confluence in serum-containing medium; and serially passage the cultured cell population for no more than about seven serial passages, wherein the cultured cells are seeded at a low density at each passage.

To select optimal culture conditions for isolating bone marrow cell populations that can treat neurodegeneration, cell populations were initially grown under different culture conditions, such as cell density at seeding, cell passage number, medium composition, or cell separation method (see Table 1).

In the presence of α-MEM supplemented with 2mM GlutaMax (Invitrogen) and 10% fetal bovine serum (FBS, HyClone or GIBCO) or α-MEM supplemented with 2mM GlutaMax (Invitrogen) and 10% fetal bovine serum (FBS, HyClone or GIBCO) (Mediatech), bone marrow cells from untreated bone marrow or after density separation or ACK lysis are seeded on tissue/cell culture plastic. After washing to remove unattached cells, adhered cells are allowed to proliferate to near confluence. The cells are then serially passaged for a total of 3, 4, 5 or 6 passages.

The candidate bone marrow cell populations were then tested for their ability to treat neurodegeneration in both in vitro OGD assays and in vivo MCAO studies.

Table I

cell Cellular part culture medium Passaging Cell 1 Untreated BM* αMEM + 10% FBS 3 Cell 2 Using Ficoll αMEM + 10% FBS 3 Cell 3 Use ACK lysis buffer αMEM + 10% FBS 3 Cell 4 Using Ficoll Serum-free medium (StemPro) 6 Cell 5 Use ACK lysis buffer Serum-free medium (StemPro) 5 Cell 6 Using Ficoll Serum-free medium (StemPro) 4 Cell 7 Use ACK lysis buffer Serum-free medium (StemPro) 4

*marrow

2) Primary screening using an in vitro oxygen/glucose deprivation (OGD) protocol

An in vitro oxygen/glucose deprivation (OGD) protocol was used to evaluate different parameters of the production methods outlined above, such as bone marrow preparation with or without density separation, seeding density, passage number, culture medium, and/or combinations thereof, to determine the optimal procedure for isolating candidate bone marrow cell populations for the treatment of neurodegeneration.

The in vitro OGD model was chosen for initial screening because it mimics the neurodegeneration caused by ischemic stroke. Specifically, OGD tests whether specific candidate myeloid cell subsets can prevent neuronal cell death in culture and induce the secretion of neuroprotective trophic factors, such as bFGF and IL-6.

In an in vitro oxygen-glucose deprivation (OGD) model, primary mixed cultures of rat neurons and astrocytes (at a 1:1 ratio) were exposed to an OGD insult (8% oxygen; Earle's balanced salt solution without glucose) for 90 minutes and returned to physiological conditions for 2 hours, after which candidate bone marrow cell populations were added to the OGD-treated neuron-astrocyte co-cultures for an additional 3 hours. Neuronal cell viability was assessed immediately after OGD and 5 hours after OGD using standard trypan blue staining and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) methods.

Cell culture

Primary mixed cultures of rat neurons and astrocytes were maintained in culture according to the supplier's protocol (CAMBREX, MD). Immediately after thawing, cells ( ^4x104 cells/well) were seeded in 96-well plates coated with polylysine and grown in neurobasal medium (GIBCO, CA) containing 2mM L-glutamate, 2% B27 (GIBCO, CA) and 50U/ml penicillin and streptomycin in a humidified atmosphere containing 5% _CO2 at 37°C for 7-10 days. The purity of the neuronal and astrocyte populations was then assessed using MAP2 and GFAP immunostaining, respectively, and was found to be greater than 99%.

Oxygen-glucose deprivation (OGD) and co-culture with candidate bone marrow cell populations

Cultured cells were exposed to the OGD injury model as previously described, but with slight modifications (Malagelada et al., Stroke (2004) 35(10): 2396-2401). Briefly, the culture medium was replaced with glucose-free Earle's balanced salt solution (BSS) having the following composition: 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, _{1 mM NaH₂PO₄} , 26.2 _mM NaHCO₃, 0.01 mM glycine, 1.8 mM _CaCl₂ , and pH adjusted to 7.4. The cultured cells were placed in a humidified chamber to equilibrate for 15 minutes using a continuous flow of 92% _N₂ and 8% _O₂ . After equilibration, the chamber was sealed and placed in a 37°C incubator for 90 minutes. After this period, OGD is terminated by adding glucose to the culture medium and the culture is returned to a standard 95% O ₂ and 5% CO ₂ incubator. A 2-hour "reperfusion" is then allowed in standard culture medium under normoxic conditions, after which the candidate bone marrow (BM) cell population is added to the OGD-treated mixed neuron-glial cell culture for approximately 3 hours. The supernatant and BM cell population are then separated from the mixed culture by washing. Afterwards, cell viability and immunocytochemistry tests are performed on the cells, and the amount of secreted trophic factors is measured using a commercially available ELISA assay as described below.

Cell viability analysis

Cell viability was assessed at two time points: immediately after OGD and 5 hours after OGD (i.e., 2 hours of reperfusion plus 3 hours of treatment with the selected bone marrow cell population). For post-OGD viability analysis, supernatant containing bone marrow-derived cells was separated from the adherent mixed neural cell culture. Trypan blue staining was performed and the average number of viable cells was calculated in three randomly selected areas (0.2 mm ² ) in each well (n = 5 per treatment condition) to demonstrate cell viability for each treatment condition. In addition, trypan blue staining was performed on a subpopulation of bone marrow-derived cells harvested as a pellet from the supernatant.

ELISA analysis

It is hypothesized that trophic factors secreted by bone marrow-derived cells, such as bFGF and IL-6, and possibly neurotrophic factors, are involved in the treatment of neurodegeneration mimicked by OGD culture conditions. Therefore, measuring the amount of these molecules secreted into the culture medium provides a criterion for evaluating candidate bone marrow cell populations for in vivo neurodegeneration treatment. Supernatants from co-cultures of neural cells and candidate bone marrow cell populations, either under standard culture conditions or exposed to OGD, were collected and analyzed for the presence of trophic factor secretion using commercially available ELISA kits according to the manufacturer's instructions.

Figures 1A and 1B show the results of OGD analysis of bone marrow-derived cell populations treated according to the parameters described in Table I above.

Based on the results shown in Figures 1A and 1B, αMEM + 10% FBS was selected as the optimal cell culture medium. Untreated bone marrow was found to be superior to bone marrow treated with density separation methods (e.g., Ficoll-Paque or Percoll) or ACK lysis buffer. The optimal number of passages for untreated bone marrow cells in αMEM + 10% FBS was found to be no more than seven. 3) Secondary screening and selection of candidate bone marrow cell populations using an in vivo middle cerebral artery occlusion rat model.

Based on the results obtained from the initial screening in the in vitro OGD model, the bioactivity of each candidate bone marrow cell population for treating neurodegeneration was evaluated by comparing neurological deficits and infarct volumes in MCAO rats treated with the candidate bone marrow cell population with those in MCAO rats treated with saline solution alone.

Middle cerebral artery occlusion (MCAO) surgery

The animal is anesthetized with isoflurane (1.5% to 2.5% in oxygen). The scalp is shaved and wiped with alcohol and chlorhexidine surgical wipes. The animal is then placed in a stereotaxic apparatus. Starting slightly posterior to the eyes, a midline sagittal incision approximately 2.5 cm long is made, and the skull region is exposed using the rounded end of a surgical spatula. Baseline (i.e., before stroke surgery) laser Doppler recordings are obtained from the following coordinates (AP: +2.0, ML: ±2.0), using the bregma as a reference point. The skin on the ventral neck from the mandible to the manubrium is shaved and wiped with alcohol and chlorhexidine surgical wipes. The animal is then moved under a surgical microscope. A skin incision is made over the right carotid artery. The external carotid artery is isolated and ligated as distally as possible. The occipital artery is cauterized. It may also be necessary to cauterize one or two additional branches extending from the external carotid artery. A second ligature is placed proximal to the external carotid artery, and the external carotid artery is then transected between the ligatures. The pterygoid artery is ligated. After this, a temporary suture is placed around the common carotid artery to provide tension and limit blood flow. The proximal end of the external carotid artery is pulled back using a ligature, effectively straightening the carotid bifurcation. A pair of microscissors is used to make an incision at the end (stump) of the external carotid artery, and a 4-0 nylon thread with a prefabricated end is inserted into the internal carotid artery and passed upward until resistance is felt (approximately 15-17 mm). This effectively blocks the middle cerebral artery (MCA). The thread is fixed in place using a ligature around the proximal end of the external carotid artery. The contralateral common carotid artery is isolated and secured using a temporary ligature. The skin incision is closed using staples. The animal is then fixed to a stereotaxic apparatus to perform laser Doppler recording to show successful MCA occlusion. After 5 minutes, the ligature of the contralateral common carotid artery is removed. Isoflurane is interrupted, and the animal is placed in a recovery cage on an electric blanket. After 60 minutes, the animal is anesthetized again with isoflurane, and the incision is opened to test in a temporary model. The occlusive thread was removed and the end of the external carotid artery near the carotid bifurcation was ligated. The skin incision was closed using staples. The animal was again fixed in a stereotaxic apparatus to perform laser Doppler recording to demonstrate successful reperfusion. Finally, the animal was placed in a recovery cage on an electric blanket.

Neurological function tests

Each rat was subjected to the well-known modified Bederson neurological test, which involves obtaining scores from each rat for:

Contralateral hindlimb withdrawal, which measures the animal's ability to reposition the hindlimb after the hindlimb has been moved 2-3 cm laterally, and is rated from 0 (immediate reposition) to 3 (no reposition);

Beam-walking ability, rated from 0 for rats that easily cross a 2.4 cm wide, 80 cm long beam to 3 for rats that cannot stay on the beam for 10 seconds; and

• Bilateral forepaw grasp, which measures the ability to hold onto a 2 mm diameter steel rod, is rated from 0 for rats with normal forepaw grasping behavior to 3 for rats that are unable to grasp with their forepaws.

On each assessment day, the scores of all three tests were assessed over a period of approximately 15 minutes. The average score of the three tests was calculated to provide a composite neurological impairment score ranging from 0 (normal neurological function) to a maximum of 3 (severe neurological impairment). Therefore, the higher the score, the greater the neurological impairment.

Based on preliminary studies, a score above approximately 2.5 indicates that an animal has neurological deficits characteristic of a stroke.

Histology

Brain slice preparation

Brain slice preparation was performed to identify the area of brain injury. Seven or 28 days after MCA occlusion, rats were euthanized and perfused transcardially with saline followed by 4% paraformaldehyde. The brains were then fixed in 4% paraformaldehyde and then immersed in 25% sucrose. Each forebrain was sliced into 30 μm-thick coronal tissue sections with anteroposterior coordinates corresponding to bregma 5.2 mm to bregma −8.8 mm for each animal.

Measuring infarct volume

At least four coronal tissue sections per brain were processed for hematoxylin and eosin (H&E) or Nissl staining. Brain infarction was visualized using indirect lesion area, in which the intact area of the ipsilateral hemisphere was subtracted from the area of the contralateral hemisphere.

Lesion volume is expressed as the percentage of the volume of the lesion compared to the contralateral hemisphere. Histological determination of lesion volume was performed using hematoxylin and eosin (H&E) or Nissl staining, and representative images were digitally captured and processed using NIH Image J software, and quantitative image analysis was performed. Lesion volume was determined according to the following formula:

Slice thickness × sum of infarct areas in all brain slices.

To minimize artifacts in infarct size due to post-ischemic edema, infarct size in the ipsilateral hemisphere was measured indirectly by subtracting the non-infarcted area in the ipsilateral hemisphere from the total uninjured area in the contralateral hemisphere.

Measuring cell survival in the ischemic peripheral infarct zone

Randomly selected high-power fields corresponding to the infarcted area of the cortex were used to count the surviving cells in the ischemic area (see Yasuhara et al., Stem Cells and Dev, 2009). To estimate the viability of host neural cells in the ischemic cortical area, crystal violet solution (Sigma, St. Louis, MO) was used for Nissl staining, and a camera (Carl Zeiss, Axiophot2) was used to capture randomly selected fields of view in the cortical area of 3 sections and the corresponding contralateral undamaged cortex. The number of cells was determined by counting cells in randomly selected high-power fields (28,800 μm ² ). The percentage of neurons preserved in the damaged cortex relative to the undamaged side was calculated and used for statistical analysis. The brain sections were blind-coded and the total number of stained cells counted was corrected using the Abercrombie formula.

Screening candidate bone marrow cell populations using the MCAO stroke animal model

Three (for IV administration) or six (for ICA administration) male rats/group were subjected to a 1-hour transient MCAO and then injected with 1 ml of injection medium containing saline or 7.5 x 10 ⁶ bone marrow-derived cells (referred to as NCS-01 cell population) isolated according to the above protocol. Animals were followed up for up to 7 days after cell administration.

Figures 1C and 1D show that NCS-01 bone marrow cell populations administered IV or ICA provide substantial neurological or pathological benefits when administered to rats with transient MCAO. Furthermore, NCS-01 prevents host cell death by treating ischemia-induced neurodegeneration, thereby reducing infarct volume and improving neurological deficits.

The primary in vitro screening and secondary in vivo screening procedures were repeated until the method reliably and reproducibly produced an optimal bone marrow-derived cell subpopulation capable of treating neurodegeneration (referred to as the NCS-01 cell population).

The optimized NCS-01 cell population was tested again in an in vitro OGD model to confirm its anti-neurodegenerative activity in co-culture with human neurons and astrocytes (see Figure 1E), and again in vivo in a rat MCAO model (see Figure 1F). The experiment described in Figure 1F also shows that the ability of the NCS-01 cell population to treat neurodegeneration is dose-dependent.

Example 2: Standardized production procedures for generating NCS-01 cell populations capable of treating neurodegeneration

Untreated whole bone marrow is harvested from mammals that have not been pretreated with any antimitotic or antimetabolite agents such as 5-fluorouracil (5-FU). Since the bone marrow is not processed by density separation or ACK lysis, the starting whole bone marrow cell population can include both hematopoietic and non-hematopoietic cells as well as nucleated bone marrow cells and non-nucleated bone marrow cells.

Untreated bone marrow was diluted and seeded at a low density (10 ⁵ to 10 ⁶ cells/cm ² ) onto tissue/cell culture plastic surfaces and cultured in serum-containing medium (α-MEM (phenol red-free) supplemented with 2 mM GlutaMax (Invitrogen) and 10% fetal bovine serum (FBS, HyClone or GIBCO) or α-MEM (Mediatech) supplemented with 2 mM GlutaMax (Invitrogen) and 10% fetal bovine serum (FBS, HyClone or GIBCO). The cell cultures were then incubated at 37°C, 5% CO ₂ , and 80% relative humidity for 72 hours.

Cells were rinsed with D-PBS to remove non-adherent cells and RBCs from the cell culture plastic, followed by replacement of the supplemented α-MEM with complete medium, which was used for all subsequent feedings. Cell cultures (passage 0 or p0) were then incubated at 37°C, 5% _CO2 , 80% RH.

For the first passage, cells were rinsed with D-PBS and detached from the plastic using a cell detachment agent. Detached cells were harvested by centrifugation at 300 g (~1000 rpm) for 8-10 minutes. The pelleted cells were resuspended in supplemented α-MEM and seeded onto tissue/cell culture plastic surfaces at a density of approximately 750 cells/ ^cm2 .

Cells were cultured to near confluence before additional passaging.

The cells were harvested and seeded again as described above at a density of approximately 750 cells/ ^cm2 on tissue/cell culture plastic surfaces for passage 2.

For the third passage, cells were harvested using a cell separation agent and centrifugation as described above. The pelleted cells were then resuspended and concentrated. The cells were resuspended in refrigerated storage medium and 1 ml of the cell suspension was dispensed into cryovials (Nunc). Vials were frozen using a controlled speed freezer and, once the freezing process was complete, the vials were moved onto dry ice for permanent storage in a vapor phase liquid nitrogen freezer.

The vial containing cells at passage 3 (p3) constituted the MCB.

Thaw one MCB vial and seed reconstituted cells (passage 4 or p4) at a density of approximately 750 cells/ ^cm2 in α-MEM supplemented with 10% FBS and GlutaMAX ^™ onto tissue/cell culture plastic. The cells were then cultured at 37°C, 5% _CO2 , 80% RH.

For passage 4, cells were cultured to near confluence before additional passages. Cells were harvested and plated onto new tissue/cell culture plastic surfaces as described above.

For passage 5, cells were harvested and frozen as described above for MCB. Cells aliquoted into vials and cryopreserved at passage 5 (p5) constituted WCB.

When needed, thaw one WCB vial and seed the reconstituted cells on tissue/cell culture plastic at a density of approximately 750 cells/ ^cm2 in α-MEM supplemented with 10% FBS and GlutaMAX ^™ . The cells were then cultured at 37°C, 5% _CO2 , 80% RH.

For passage 6, cells were cultured to near confluence before additional passaging. Cells were harvested and plated onto new tissue/cell culture plastic surfaces as described above.

The medium was changed midway through passages (WCB to passage 6 and passages 6 and 7).

For passage 7, cells were harvested and frozen according to the method described above for MCB. Example 3: Use of NCS-01 cells to treat neurodegeneration caused by permanent versus transient middle cerebral artery occlusion (MCAO)

The infarct volume and neurological function of rats with permanent MCAO treated with NCS-01 were compared with those of rats with transient (60 min) MCAO treated with NCS-01.

Transient MCAO mimics the current standard clinical procedures used to treat arterial blockage caused by stroke, which restore blood flow to the stroke penumbra. These procedures include the administration of thrombolytics and procedures involving mechanical clot removal, such as angioplasty and/or vascular stenting.

Groups of 3 or 6 rats were subjected to permanent or transient MCAO (using reperfusion). 1 ml of saline or 1 ml of 7.5 x 10 ⁶ NCS-01 cells were then administered ICA 24 hours after ischemia, and the rats were monitored until 28 days. Neurological function was then assessed using a modified Bederson neurological test. The transient occlusion model simulates the situation of stroke patients who have been treated with tPA or have undergone clot removal.

Figure 2 shows the significant histological benefits (infarct volume; Panel A) and clinical benefits (modified Bederson scale; Panel B) of treatment with NCS-01 cell populations in two MCAO paradigms. The benefits in the temporary occlusion model were 2- to 3-fold higher than those in the permanent occlusion model. The time course of neurological responses (Panel B) showed stable improvements up to 28 days after infarction, during which time, untreated controls without reperfusion (permanent occlusion) improved by an average of 11%, while animals treated with reperfusion (temporary occlusion) and NCS-01 improved by an average of 67%.

Unexpectedly, NCS-01 was more effective in treating symptoms in the transient occlusion model, suggesting that the greatest effect is achieved when NCS-01 is added in conjunction with clot removal, either after administration of a thrombolytic agent or after removal of the clot using a mechanical device.

Example 4: Comparison between NCS-01 cells and other bone marrow-derived cells

1) Isolation of bone marrow cell populations according to Li et al.

Bone marrow cell populations (hereinafter referred to as Li bone marrow cell populations) were prepared as described in the publication by Li et al. (Journal of Cerebral Blood Flow and Metabolism (2000) 20: 1311-1319).

Primary cultured bone marrow cells were obtained from adult mice that received an intraperitoneal injection of the antimetabolite drug 5-fluorouracil (5-FU, 150 mg/kg) 2 days prior to harvest (Randall and Weissman, 1997). Fresh whole bone marrow was aseptically harvested from the tibia and femur using a 21-gauge needle connected to a 1 mL syringe containing phosphate-buffered saline (PBS, 0.5 mL). The bone marrow was mechanically dissected until a milky, homogenous, single-cell suspension was formed. Red blood cells were removed from the bone marrow using 0.84% _NH4Cl , and the number of nucleated bone marrow cells was determined using a hemocytometer. ² x 106 nucleated cells were seeded in tissue culture flasks in Iscove's modified Dulbecco's medium supplemented with fetal bovine serum (10%). After 3 days of incubation, the cells adhered tightly to the plastic and were resuspended in fresh Iscove's modified Dulbecco's medium in a new flask and grown for three additional passages.

2) Comparison of Li cell populations with NCS-01 cell populations in in vitro OGD assays

Two batches of NCS-01 were manufactured from the same MCB and different WCBs of bone marrow and tested together with the Li cell population in the in vitro OGD model outlined in Figure 3.

As shown in Figure 4, both batches of NCS-01 cells produced the same increase in the secretion of both IL-6 and bFGF.Thus, the NCS-01 cell populations generated using the optimized production procedure described above consistently treated neurodegeneration in the in vitro OGD assay.

In contrast, Li cell populations correctly isolated as described in the Li (2002) publication produced 4-5 fold less bFGF and IL-6 than NCS-01 cell populations in the OGD assay.

2) Comparison of Li cell populations with NCS-01 cell populations in in vivo MCAO analysis

The ability of NCS-01 and Li cell populations to treat neurodegeneration in vivo was tested using a MCAO rat model. Figures 5A and 5B show the effects of the cells on host cell viability, infarct volume, and neurological deficits. Consistent with the above studies, the NCS-01 cell population prevented host cell death (see Figure 5A) by treating ischemia-induced neurodegeneration, thereby reducing infarct volume and improving neurological deficits (see Figure 5B).

In contrast, the Li cell population failed to show any statistically significant activity in infarct volume or neurological function. Thus, these data confirm that the ability of the NCS-01 population to treat neurodegeneration is limited by the production method and that the NCS-01 cell population is different from the cell population described in Li's 2000 publication.

Any patent, patent application, publication or other public material mentioned in this specification is hereby incorporated by reference in its entirety. If any material or portion thereof incorporated by reference conflicts with existing definitions, statements or other public materials described herein, it is incorporated only to the extent that no conflict arises between the incorporated material and the present disclosure.

Claims (7)

1. A kit comprising a thrombolytic agent and a heterogeneous subset of bone marrow cells for treating neurodegeneration caused by ischemia. The heterogeneous bone marrow cell subsets mentioned above are prepared by a method comprising the following steps: a) Provide a population of bone marrow cells in the form of unprocessed human bone marrow; b) The bone marrow cell population was seeded on a plastic surface at a low density of ^10⁵ cells/ ^cm² to ^10⁶ cells/ ^cm² ; c) Wash the inoculated cell population to remove unattached cells; d) Culture attached cells from the washed cell population to confluence in a serum-containing medium; e) The cultured cells are passaged continuously in the serum-containing medium for no more than seven consecutive passages, wherein at each passage, the cultured cells are seeded at a low density of 750 cells/ ^cm² ; and f) Obtain the heterogeneous bone marrow cell subsets; The heterogeneous myeloid cell subsets mentioned therein treat the neurodegeneration caused by ischemia. 2. The kit according to claim 1, wherein the thrombolytic agent is at least one selected from streptokinase, urokinase, and recombinant tissue plasminogen activator. 3. The kit according to claim 2, wherein the recombinant tissue plasminogen activator is alteplase, reteplase, or tenecteplase. 4. A kit comprising a heterogeneous subset of bone marrow cells for treating neurodegeneration caused by ischemia and a tool for mechanically removing blood clots. The heterogeneous bone marrow cell subsets mentioned above are prepared by a method comprising the following steps: a) Obtaining bone marrow cell populations from untreated bone marrow; b) The bone marrow cell population was seeded on a plastic surface at a low density of ^10⁵ cells/ ^cm² to ^10⁶ cells/ ^cm² ; c) Wash the inoculated cell population to remove unattached cells; d) Culture attached cells from the washed cell population to confluence in a serum-containing medium; e) The cultured cells are passaged continuously in the serum-containing medium for no more than seven consecutive passages, wherein at each passage, the cultured cells are seeded at a low density of 750 cells/ ^cm² ; and f) Obtain the heterogeneous bone marrow cell subsets; The heterogeneous myeloid cell subsets mentioned therein treat the neurodegeneration caused by ischemia. 5. A composition for treating ischemic neurodegeneration, comprising a thrombolytic agent, a heterogeneous subset of bone marrow cells for treating ischemic neurodegeneration, and a carrier for injecting the composition. The heterogeneous myeloid cell subsets mentioned above are prepared by a method comprising the following steps: a) Obtaining bone marrow cell populations from untreated bone marrow; b) The bone marrow cell population was seeded on a plastic surface at a low density of ^10⁵ cells/ ^cm² to ^10⁶ cells/ ^cm² ; c) Wash the inoculated cell population to remove unattached cells; d) Culture attached cells from the washed cell population to confluence in a serum-containing medium; e) The cultured cells are passaged continuously in the serum-containing medium for no more than seven consecutive passages, wherein at each passage, the cultured cells are seeded at a low density of 750 cells/ ^cm² ; and f) Obtain the heterogeneous bone marrow cell subsets; The heterogeneous myeloid cell subsets mentioned therein treat the neurodegeneration caused by ischemia. 6. The composition according to claim 5, wherein the thrombolytic agent is at least one selected from streptokinase, urokinase, and recombinant tissue plasminogen activator. 7. The composition according to claim 6, wherein the recombinant tissue plasminogen activator is alteplase, reteplase, or tenecteplase.