JP6438527B2 - Cell therapy for the treatment of neurodegeneration - Google Patents

Description

  [1] This disclosure describes cell compositions and methods for the treatment of neurodegeneration.

  [2] Neurodegeneration is a pathological condition that results in the death of nervous system cells. The causes of neurodegeneration can vary and are not always definitive, but many neuropathies share neurodegeneration as a common pathological condition. For example, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS) all result in chronic neurodegeneration, which is characterized by slow progressive nervous system cell death over several years; In contrast, acute neurodegeneration is the result of axonal transection as a result of trauma such as ischemia or traumatic brain injury such as stroke or due to demyelination or trauma caused by, for example, spinal cord injury or multiple sclerosis. As characterized by the sudden onset of neural cell death.

  [3] Neurodegeneration can also be induced by a variety of neuronal cell injuries resulting, for example, from alcoholism, drug addiction, neurotoxin and radiation exposure. Evidence for neurodegeneration can be seen in dementia, epilepsy, various mental disorders, and even as part of the normal aging process.

  [4] Regardless of the underlying cause, once neurodegeneration is induced, the consequences of all these disorders are always the same-ultimately the death of the nervous system cells, and increasing amounts of The evidence points out.

  [5] Stroke is accompanied by acute neurodegeneration (loss of function associated with rapid loss of nervous system cells in the central nervous system) due to occlusion (cerebral infarction) or rupture (bleeding) of blood vessels leading to or within the brain. It is usually emergency care because it can cause permanent nerve damage, systemic complications, and even death. Stroke is the leading cause of disability in adults in the United States and Europe and the second leading cause of death worldwide. Stroke is more than 1 in 15 deaths in the United States and is the third most common cause of death after heart disease and cancer (American Heart Association. Heart Disease and Stroke Statistics-2009 Update. Dallas, Texas: 2009; Rosamond et al. Heart disease and stroke statistics-2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 2007; 115: e69-e171). All third strokes are fatal (Haheim et al. Risk factors of stroke incidence and mortality. A 12-year follow-up of the Oslo Study. Stroke. 1993; 24 (10): 1484-9) . About 6% of all deaths before age 65 and about 10% of all deaths after that are due to stroke (Donnan et al. Stroke. Lancet. 2008; 371 (9624): 1612-23 ). Thus, statistical data demonstrate that severe disability occurs in those who have experienced many unfortunately frequent strokes. In fact, stroke is the leading cause of in-patient Medicare reimbursement for long-term adult care. The total costs associated with stroke treatment and rehabilitation currently exceed $ 45 billion annually and will undoubtedly continue to be involved in the overall increase in health costs in the United States and other major developed countries.

  [6] Cerebral infarction is the most common form of stroke, accounting for over 80 percent of all strokes. They arise from arterial blockage, usually due to thrombus formation or less but due to embolism. Onset is generally abrupt, followed by local neurological deficits. About 20% of patients will die within a few days, especially if the infarction is significant. An additional 10% of patients die within a few weeks of the first stroke. Unfortunately, survivors usually have severe disabilities. Depending on the area of the brain that has been damaged, symptoms include weakness and sensory disturbances on one side of the face or limbs, and cognitive and language impairments. The larger the area of the damaged brain, the more functions can be impaired. Some improvement in functionality may begin within a few days and generally recovers over months. Nevertheless, the extent of recovery is unpredictable and is generally incomplete. According to the American Stroke Association, 15-30% of seizure survivors have permanent disability and 20% require institutional care for 3 months after onset (Harmsen et al. Long-term risk factors for stroke: twenty-eight years of follow-up of 7457 middle-aged men in Goeteborg, Sweden. Stroke. 2006; 37 (7): 1663-7).

  [7] Cerebral infarction is a central lesion where nerve cells die within minutes of oxygen deprivation and the surrounding penumbra (which receives some blood flow and hence some oxygen but is not normal) To. Cell death is slower in the ischemic penumbra, generally progressing over several hours, caused by variable oxygen deprivation, toxicities generated by the ischemic cascade, and glutamate release in the central lesion. Thus, current area interventions are primarily aimed at reducing the injury status of the stroke penumbra.

  [8] However, there are still limited treatments for acute cerebral infarction.

  [9] Because brain damage occurs as a result of decreased blood flow to the brain, current therapies remove arterial blockage by either clot lysis (thrombolysis) or clot mechanical removal (thrombotomy). Try. The faster the blood flow is restored, the less brain cell death and the greater the chance of avoiding permanent sequelae.

  [10] Currently, there are only two FDA approved treatments for stroke in the United States:

  • Recombinant tissue plasminogen activator (rt-PA; Genentech), a drug that lyses arterial clots; and • Merci Retrieval System (Concentric Medical Inc.) and Penumbra System P ), That is, an instrument for mechanically removing blood clots.

  [11] All the above therapies have significant limitations.

  [12] To be effective, thrombolytic therapy must be administered within 3 to 4.5 hours of symptom onset, which is effective in about 3% of patients with acute cerebral infarction. It means that you can only receive rt-PA therapy. Furthermore, thrombolytic therapy is substantially associated with an increased risk of cerebral hemorrhage, which further limits its use in certain individuals.

American Heart Association. Heart Disease and Stroke Statistics-2009 Update. Dallas, Texas: American Heart Association; 2009 Rosamond et al. Heart disease and stroke statistics-2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 2007; 115: e69-e171 Haheim et al. Risk factors of stroke incidence and mortality. A 12-year follow-up of the Oslo Study. Stroke. 1993; 24 (10): 1484-9 Donnan et al. Stroke. Lancet. 2008; 371 (9624): 1612-23 Harmsen et al. Long-term risk factors for stroke: twenty-eight years of follow-up of 7457 middle-aged men in Goeteborg, Sweden. Stroke. 2006; 37 (7): 1663-7

  [13] For these reasons, there is an unmet need in the art for safe and effective therapies that alleviate and / or prevent neurodegeneration, particularly neurodegeneration caused by ischemia.

[14] Accordingly, in one aspect, the present invention provides:
a) collecting a population of bone marrow cells from untreated bone marrow;
b) seeding the population of bone marrow cells at a low density onto a plastic surface;
c) washing the seeded cell population to remove non-adherent cells;
d) culturing the adherent cell population to near confluence in serum-containing medium;
e) continuously culturing a population of cultured cells at about 7 consecutive passages or less, seeding the cultured cells at low density at each passage;
f) a heterogeneous subpopulation of BM cells prepared by a method comprising collecting a subpopulation of heterogeneous bone marrow cells, comprising:
An effective amount provides a heterogeneous bone marrow cell subpopulation that is effective in the treatment of neurodegeneration.

  [15] In one aspect, an effective amount of a heterogeneous bone marrow cell subpopulation is effective in treating neurodegeneration caused by ischemia.

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

[17] In one aspect, a population of bone marrow cells is seeded at a density of about 10 ² to 10 ⁶ cells / cm ² .

[18] In one aspect, cultured cells are seeded at a density of about 750 cells / cm ² or less during passage.

  [19] Untreated bone marrow is taken from subjects that have not been pretreated with an anti-tumor agent such as 5-fluorouracil, including any agent that modulates cell division, for example, an antimetabolite used for chemotherapy. it can.

  [20] In one aspect, a heterogeneous bone marrow cell subpopulation cannot be separated by density fractionation, such as Ficoll ™ or Percoll ™ gradient, or ACK (Ammonium-Chloride-Potassium) lysis .

  [21] In another aspect of the invention, a heterogeneous bone marrow cell subpopulation is further tested in an experimental model of neurodegeneration caused by ischemia, and cells exhibiting the ability to treat neurodegeneration caused by ischemia Select only the population. The experimental model of neurodegeneration caused by ischemia may be an oxygen / glucose deprivation (OGD) cell culture model or an animal model of stroke. In certain aspects, the experimental model may be an oxygen / glucose depletion (OGD) cell culture model followed by an animal model of stroke.

  [22] In another aspect, after injection into the bloodstream, cells within the heterogeneous subpopulation migrate to the site of neurodegeneration.

  [23] In one aspect, the neurodegeneration caused by ischemia is cerebral infarction.

  [24] In another embodiment, the present invention also provides a method for treating neurodegeneration caused by ischemia, wherein the heterogeneous bone marrow cell subpopulation is injected into the bloodstream of a mammal with neurodegeneration. And injecting a heterogeneous bone marrow cell subpopulation provides a method of reducing neuronal deficits caused by neurodegeneration.

  [25] If the neurodegeneration is caused by a cerebral infarction, the method can further include additional treatment to increase blood flow through the occluded blood vessel, and heterogeneous bone marrow combined with the additional treatment. By injection of a subpopulation of cells, the neurological deficit is significantly reduced after either an additional treatment alone or an injection of a heterogeneous bone marrow cell subpopulation without additional treatment.

  [26] In one aspect, additional treatment to increase blood flow through the occluded blood vessel is performed concurrently with infusion of a heterogeneous bone marrow cell subpopulation.

  [27] In another aspect, additional treatment to increase blood flow through the occluded blood vessel is performed prior to infusion of the heterogeneous bone marrow cell subpopulation.

  [28] When neurodegeneration occurs due to cerebral infarction, the blood vessels may be occluded by blood clots. A clot can result from the rupture of an atherosclerotic plaque.

  [29] If neurodegeneration is caused by a cerebral infarction, additional treatment to increase blood flow through the occluded blood vessel can include administration of 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.

  [30] In other aspects, if the neurodegeneration is caused by a cerebral infarction, additional treatment to increase blood flow through the occluded blood vessel can include mechanical clot removal from the occluded blood vessel.

  [31] In yet another aspect, the clot can be captured with a filter.

  [32] In other aspects, if neurodegeneration is caused by a cerebral infarction, additional treatment to increase blood flow through the occluded blood vessel can include performing angioplasty and / or vascular stenting .

  [33] A heterogeneous subpopulation of bone marrow cells can be injected intravenously, intraarterially, for example, into the carotid artery, or into the brain.

  [34] In another embodiment, the present invention also provides a method for treating neurodegeneration caused by ischemia, wherein a heterogeneous bone marrow cell subpopulation is treated with mammalian blood with ischemia-induced neurodegeneration. A method of reducing the volume of infarcts caused by ischemia by injecting a subpopulation of bone marrow cells.

  [35] If the neurodegeneration is caused by a cerebral infarction, the method can further include additional treatment to increase blood flow through the occluded blood vessel, and heterogeneous bone marrow combined with the additional treatment The injection of a subpopulation of cells significantly reduces the infarct volume after either an additional treatment alone or an injection of a heterogeneous bone marrow cell subpopulation without additional treatment.

  [36] In one aspect, if neurodegeneration is caused by cerebral infarction, additional treatment to increase blood flow through the occluded blood vessel includes administration of a thrombolytic agent. The thrombolytic agent may be at least one of streptokinase, urokinase, and recombinant tissue plasminogen activator. Further, the recombinant tissue plasminogen activator may be alteplase, reteplase, desmoteplase or tenecteplase.

  [37] In other aspects, if the neurodegeneration is caused by a cerebral infarction, additional treatment to increase blood flow through the occluded blood vessel can include performing angioplasty and / or stent insertion.

  [38] In other aspects, if the neurodegeneration is caused by a cerebral infarction, additional treatment to increase blood flow through the occluded blood vessel may include mechanical removal of a clot near the infarct site.

  [39] According to the second method, a heterogeneous subpopulation of bone marrow cells can be injected intravenously, intraarterially, eg, into the carotid artery, or into the brain.

  [40] When neurodegeneration is caused by cerebral infarction, the present invention can also provide a kit comprising a thrombolytic agent and the heterogeneous bone marrow cell subpopulation. The thrombolytic agent may be at least one of streptokinase, urokinase, and recombinant tissue plasminogen activator. Further, the recombinant tissue plasminogen activator may be alteplase, reteplase, or tenecteplase.

  [41] When the neurodegeneration is caused by cerebral infarction, the present invention can further provide a kit comprising a means for mechanically removing the heterogeneous bone marrow cell subpopulation and the blood clot.

  [42] Further, when neurodegeneration is caused by cerebral infarction, the present invention may be caused by ischemia comprising a thrombolytic agent, said heterogeneous bone marrow cell subpopulation, and a carrier for injecting the composition. A composition for treating neurodegeneration can be provided. The thrombolytic agent may be at least one of streptokinase, urokinase, and recombinant tissue plasminogen activator. Further, the recombinant tissue plasminogen activator may be alteplase, reteplase, or tenecteplase.

[43] The present invention also provides a method of preparing a heterogeneous bone marrow cell subpopulation for the treatment of neurodegeneration comprising:
a) collecting a heterogeneous population of bone marrow cells from untreated bone marrow;
b) seeding the heterogeneous bone marrow cell population on a plastic surface at low density;
c) washing the seeded cell population to remove non-adherent cells;
d) culturing adherent cells from the washed cell population to near confluence in serum-containing medium;
e) serially culturing each population of cultured cells in about 7 consecutive passages or less, seeding the cultured cells at each density at a low density, thereby obtaining a heterogeneous subpopulation of bone marrow cells;
A method comprising:

  [44] In one aspect, the untreated bone marrow is pretreated with any agent that modulates cell division, for example, an anti-tumor agent such as 5-fluorouracil, including an antimetabolite used in chemotherapy. Can be collected from untargeted subjects.

  [45] In some aspects, a heterogeneous subpopulation of bone marrow cells cannot be separated by density fractionation, such as Ficoll ™ or Percoll ™ gradient, or ACK (potassium ammonium chloride) lysis.

  [46] In another aspect of the invention, the heterogeneous bone marrow cell subpopulations are further tested in an experimental model of neurodegeneration caused by ischemia and exhibiting the ability to treat neurodegeneration caused by ischemia Select only the population. The experimental model of neurodegeneration caused by ischemia may be an oxygen / glucose depletion (OGD) cell culture model or an animal model of stroke. In certain aspects, the experimental model may be an oxygen / glucose depletion (OGD) cell culture model followed by an animal model of stroke. In one aspect, both models can be used to test a heterogeneous bone marrow cell subpopulation.

[47] In another aspect, the invention also provides a method of optimizing an experimental protocol for isolating a heterogeneous cell population that treats neurodegeneration caused by ischemia, comprising:
Separating the cell population according to an experimental protocol,
Treating neurodegeneration caused by ischemia in the cell population,
Optimal parameters for each stage of the protocol are determined by testing the effect each parameter has on the efficacy of isolated cell populations to treat neurodegeneration caused by ischemia in an experimental animal model of ischemia,
Can provide a method.

  [48] In certain aspects, the parameters can include cell density at seeding, cell passage number, culture medium composition, or cell fraction.

  [49] In other aspects, the experimental model of ischemia may be an oxygen / glucose depletion (OGD) cell culture model, or an animal model of stroke, such as a middle cerebral artery occlusion (MCAO) animal model. Both models can also be used. In certain aspects, the experimental model may be an oxygen / glucose depletion (OGD) cell culture model followed by an animal model of stroke.

  [50] The above aspects have a number of advantages, including the ability of heterogeneous bone marrow cell subpopulations to treat neurodegeneration caused by cerebral infarction. This cell population significantly reduces the area of infarction and improves nerve function.

  [51] In combination with thrombolytics and mechanical clot removal methods, this cell therapy ensures that there is a significant clinical effect on patient survival, functionality, and quality of life after stroke.

[52] Those skilled in the art will appreciate that the drawings described below are for illustration purposes only. The drawings are not intended to limit the teachings of the invention in any way.
[53] FIG. 1 shows the results from screening candidate subpopulations of bone marrow derived cells using in vitro OGD models (FIGS. 1A, 1B and 1E) and in vivo MCAO rat models (FIGS. 1C, 1D and 1F). [54] FIGS. 1A and 1B show host cell viability and cytokine release (bFGF and IL-6), respectively, in an in vitro OGD model in response to the presence or absence of seven different candidates of bone marrow-derived cell subpopulations. Show. FIGS. 1A and 1B show host cell viability and cytokine release (bFGF and IL-6), respectively, in an in vitro OGD model in response to the presence or absence of seven different candidates of bone marrow derived cell subpopulations. [55] FIGS. 1C and 1D show host cell viability, infarct volume and in vivo MCAO rat model in response to intravenous (IV) or intraarterial (ICA) injected NCS-01 bone marrow cell subpopulations or saline. This is a comparison of nerve function. [55] FIGS. 1C and 1D show host cell viability, infarct volume and in vivo MCAO rat model in response to intravenous (IV) or intraarterial (ICA) injected NCS-01 bone marrow cell subpopulations or saline. This is a comparison of nerve function. [56] FIG. IE shows host cell viability and cytokine release (bFGF and IL-6) in an in vitro OGD model in response to the presence or absence of NCS-01 bone marrow derived cell subpopulations. [57] FIG. 1F shows changes in infarct volume and neural 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 derived cells. [58] FIG. 2 shows changes in infarct volume in transient (60 minutes) or permanent MCAO rat models in response to ICA infusion of 1 ml of 7.5 × 10 ⁶ NCS-01 cells or saline (panel A) and changes in nerve function (Panel B; Modified Bederson Scale (measured with 0 = normal to 3 = most severe)). [59] Figure 3 shows a schematic diagram describing the protocol used for the in vitro oxygen glucose depletion (OGD) assay. [60] FIG. 4 shows the relative levels of bFGF and IL-6 cytokines secreted into the cell culture medium of the OGD assay after addition of saline, Li cells or NCS-01 cells. [61] FIGS. 5A and 5B show host cell viability, infarct volume and neural function in an in vivo MCAO rat model in response to injection of saline, Li cells and NCS-01 cells. [61] FIGS. 5A and 5B show host cell viability, infarct volume and neural function in an in vivo MCAO rat model in response to injection of saline, Li cells and NCS-01 cells.

  [62] The practice of the present invention employs general molecular biology techniques that can be accomplished by one skilled in the art unless otherwise indicated. Such techniques are well known to those skilled in the art and are fully explained in the literature. For example, Ausubel, et al., Ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including all supplements; Sambrook, et al., Molecular Cloning: See A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989).

  [63] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. This specification also provides definitions of terms to assist in interpreting the disclosure of the present application and the claims. If the definition does not match any other definition, the definition set forth in this application will control.

  [64] Bone marrow (BM) derived cells contain a heterogeneous mixture of many different types of cells. The bone marrow is composed of two major cell lines that belong to two distinct lineages—hematopoietic tissue and the accompanying supporting stroma. Thus, it is known that at least two distinct stem cells coexist in the bone marrow, namely hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) (Bianco, M. Riminucci, S. Gronthos, and PG Robey, " Bone marrow stromal stem cells: nature, biology, and potential applications, "Stem Cells, vol. 19, no. 3, pp. 180-192, 2001).

  [65] MSCs can be roughly defined by both cell surface markers and their ability to attach to tissue / cell culture plastic. Therefore, the MSC population is necessarily heterogeneous, i.e. not a cloned cell population. Even though MSC subpopulations share one or more common cell surface markers, their biological activity may nevertheless differ significantly depending on the preparation method used to isolate them . Described herein is a two-step screening protocol for identifying a heterogeneous population of bone marrow cells that is effective in treating neurodegeneration, including acute onset neurodegeneration caused by ischemia.

  [66] In a first stage, in a co-culture comprising a candidate subpopulation of bone marrow cells and neural cells, the subpopulation of bone marrow cells is screened for their ability to attenuate the effects of oxygen glucose depletion (OGD). The activity of candidate bone marrow cell subpopulations to attenuate neurodegeneration is assessed by measuring the secretion of nutrient factors (bFGF and IL6) in the culture medium and measuring cell viability in an OGD assay. .

  [67] In the second stage, the bone marrow-derived cell population with the strongest activity in the OGD assay is then further screened by testing in an in vivo MCAO rat model of neurodegeneration caused by ischemia. This screening procedure, as defined herein, facilitates the identification of a sub-population of heterogeneous bone marrow cells called NCS-01 that significantly attenuates neurodegeneration caused by ischemia.

  [68] Untreated bone marrow cells are readily available to those skilled in the art.

  [69] As used herein, the term "untreated bone marrow" refers to human bone marrow cell aspirates that have not undergone any additional processing, such as density fractionation or cell sorting.

  [70] In other embodiments, "untreated bone marrow" has been pretreated with any agent that prevents normal cell growth and division, including chemotherapeutic agents such as cytostatics or antimetabolites. Obtained from no subject.

  [71] Examples of such drugs are in Cancer Principles and Practice of Oncology, V.T. Devita and S. Hellman (Author), 6th edition (February 15, 2001), Lippincott Williams & Wilkins Publishers.

  [72] The term "antimetabolite" as used herein relates to a compound that inhibits or disrupts DNA synthesis resulting in cell death. Examples of antimetabolites include, but are not limited to: 6-mercaptopurine; cytarabine; fludarabine; flexuridine; 5-fluorouracil; capecitabine Raltitrexed; methotrexate; cladribine; gemcitabine; gemcitabine hydrochloride; thioguanine; hydroxyurea; DNA demethylating agents such as 5-azacytidine and decitabine (decitarexate) ); As well as folic acid antagonists such as, but not limited to, pemetrexed.

  [73] As used herein, the term "density fractionation" is a well-known method for fractionating bone marrow cells based on cell density using Ficoll-Paque ™ or Percoll ™ gradients. Represents laboratory operation. For example, Ficoll-Paque ™ is placed on the bottom of the conical tube, and then the untreated bone marrow is gradually laminated onto the Ficoll-Paque ™. After centrifuging the cells through a Ficoll gradient, the cells are separated into the layers from top to bottom according to density as follows: plasma and other components, a layer of mononuclear cells called the buffy coat (white blood cell layer) and the peripheral Layers containing peripheral blood mononuclear cells (PBMC) and mononuclear cells (MNC), as well as erythrocytes and granulocytes in pellets. Red blood cells are separated from PBMC by this fractionation operation. To prevent clotting, ethylenediaminetetraacetic acid (EDTA) and heparin are commonly used in conjunction with Ficoll-Paque ™.

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

[75] As used herein, the term "seeding a population of bone marrow cells at a low density" relates to the concentration of bone marrow cells added at the beginning of cell culture. In certain aspects, 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 .

  [76] As used herein, "neurodegeneration" refers to any pathological condition that results in progressive loss of neuronal cell structure or function, including the death of neural cells. Therefore, neurodegeneration is a pathological condition caused by neuropathy.

  [77] In one embodiment, the phrase "neural cell" includes neural cells (ie, neurons, such as monopolar, bipolar or multipolar neurons) and their precursors, and glia Cells (eg, macroglia cells such as oligodendrocytes, Schwann cells and astrocytes, or microglia cells) and their precursors are included.

  [78] In one embodiment, an “effective amount” is a clinical of a symptom and / or pathological condition associated with neurodegeneration, including delaying, cessation or recovery of neurodegeneration, reducing neuronal deficits, or improving neural response. Represents the optimal number of cells required to induce significant improvement. In certain embodiments, an effective amount of the NCS-01 cell population represents the optimal number of cells required to reduce the volume of infarct caused by a sudden onset of acute neurodegeneration following a stroke. An effective amount of an NCS-01 cell population appropriate for a particular organism can be determined by one skilled in the art using routine experiments.

  [79] As used herein, "treatment of neurodegeneration" refers to treating neurodegeneration with the NCS-01 cell population, resulting in delayed, arrest or recovery of neurodegeneration, neurodegeneration. Symptoms and / or pathological conditions associated with neurodegeneration are clinically significantly improved, including reduction or improvement of neural response.

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

  [81] Described below is the procedure for separating and characterizing a heterogeneous bone marrow cell subpopulation from untreated whole bone marrow that is optimal for treatment of neurodegeneration, including treatment of neurodegeneration due to ischemia.

  Isolation of candidate bone marrow cell populations effective in treating neurodegeneration caused by ischemia

  [82] Untreated whole bone marrow is taken from a mammal that has not been pretreated with a cytostatic or antimetabolite, such as 5-fluorouracil (5-FU).

[83] The untreated bone marrow cells are then grown directly on tissue / cell culture plastic and serially passaged in serum-containing medium. At each passage, the cells are seeded at a very low density, ie, about 750 cells / cm ² or less, cultured to near confluence, and then passaged further. Unattached cells are removed by washing. Since this bone marrow has not been processed by density fractionation, the starting population of whole bone marrow cells contains both hematopoietic and non-hematopoietic cells, as well as nucleated and non-nucleated bone marrow cells.

[84] Next, a master cell bank (MCB) and a working cell bank (WCB) are established and stored frozen at the third and fifth passages, respectively. If necessary, WCB cells are seeded at a very low density, eg, about 750 cells / cm ² or less, grown in serum-containing medium, then harvested using standard methods and stored frozen.

  A two-step selection protocol for assessing the ability of a population of bone marrow cells to attenuate neurodegeneration

  [85] A “candidate” population of bone marrow cells can then be screened in a two-step method that selects the optimal population of bone marrow cells for treatment of neurodegeneration.

  [86] In the first stage, a candidate population of bone marrow cells is tested in an in vitro oxygen glucose depletion assay. Here, candidates for a population of bone marrow cells are co-cultured with neural cells under experimental conditions that mimic neurodegeneration. Cell populations that have been shown to attenuate neurodegeneration in vitro are then screened for their ability to treat neurodegeneration in vivo.

  [87] In the second stage, selected bone marrow cell population candidates are evaluated in a rat MCAO model of neurodegeneration caused by ischemia. A candidate population of heterogeneous bone marrow cells that exhibits maximal activity to attenuate neurodegeneration in vivo is then selected.

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

  Identification of optimal cell culture conditions to isolate a population of bone marrow cells with activity to attenuate neurodegeneration

  [89] This two-step screening protocol identifies the optimal experimental conditions for culturing a population of bone marrow cells that can treat neurodegeneration, such as cell density at seeding, cell passage number, cell media composition, or cell fractionation method. Also useful.

[90] Using the two screening method, the optimal replated cell concentration is about 750 cells / cm ² or less at each passage. The optimal culture medium is a serum-containing medium. The NCS-01 cell population can be passaged from about 7 or about 6, or about 5, or about 4, or about 3, or about 2 passages or less from the initial plating of untreated whole bone marrow. . Long-term passages, i.e. passages beyond the first plating of untreated whole bone marrow beyond passage 7, diminish or abolish the ability of NCS-01 to treat neurodegeneration in OGD in vitro assays and MCAO rat models To do.

  [91] The OGD assay for neurodegeneration and the MCAO rat model are described in detail below.

  In vitro screening of a population of bone marrow cells that can treat neurodegeneration

  [92] Candidate bone marrow cell populations are first tested for their ability to block neurodegeneration, according to the culture conditions of oxygen-glucose depletion (OGD), which mimics neurodegeneration caused by ischemia, in neuronal-astrocyte co-culture. Screen.

  [93] Primary mixed cultures of rat or human neurons and astrocytes are first exposed to oxygen glucose depletion (OGD) culture conditions (eg, 8% oxygen, glucose-free medium) for about 0.5-3 hours. Induces neurodegeneration. The oxygen-glucose depletion of the cell culture is then stopped and the OGD-induced neural cells are cultured for 2 hours under physiological conditions and then co-cultured for an additional 3 hours in the presence of a candidate population of bone marrow cells. Neurodegeneration is then assessed by measuring host cell viability at 0 and 5 hours after OGD in the presence or absence of a candidate population of bone marrow cells. Viability of host cells (neurons and astrocytes) is assessed using, for example, trypan blue staining and / or MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay can do.

  [94] About 5%, or about 10%, or about 15% in the presence of a candidate for a population of bone marrow cells, as compared to the cell viability of a control (no candidate for a population of bone marrow cells), or An increase in cell viability of about 20%, or about 25%, or more indicates that the candidate bone marrow cell population protects and rescues neuronal / astrocyte co-cultures from neurodegeneration caused by oxygen and glucose depletion. It is an indicator of what can be done.

  [95] In addition, the culture medium of neuronal / astrocytic cell cultures subjected to OGD conditions in the presence or absence of a candidate population of bone marrow cells for induced secretion of trophic factors such as bFGF and / or IL-6 It can also be assayed with a commercially available ELISA kit.

  [96] In certain embodiments, candidates for a population of bone marrow cells are selected to increase the amount of bFGF and / or IL-6 that is secreted into the culture medium of neurons / astrocytes in response to oxygen glucose depletion. Select according to ability to induce (but not in the absence of oxygen glucose depletion).

  [97] In other embodiments, candidates for a population of bone marrow cells are at least 2 of the amount of bFGF and / or IL-6 that they secrete into the culture medium of neurons / astrocytes in response to oxygen glucose depletion. Select according to the ability to induce a fold increase or more (but not in the absence of oxygen glucose depletion).

  [98] Candidate bone marrow cell populations that reduce the incidence of OGD-induced cell death and induce increased secretion of trophic factors (eg, bFGF and / or IL-6), then in an in vivo MCAO rat model Select for screening (see below).

  [99] 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 secretion of trophic factors by at least 10% or more, You can choose for.

  In vivo screening of a candidate population of bone marrow cells that can treat neurodegeneration

  [100] Candidate bone marrow cell populations selected in the OGD assay screening step described above are tested for their ability to treat neurodegeneration in vivo. For example, candidates for a population of bone marrow cells can be tested for their ability to treat neurodegeneration in experimental animal models of neurodegeneration, including transgenic models of neurodegenerative diseases (see, for example, 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).

  [101] In one embodiment, the experimental animal model of neurodegeneration is an animal model of stroke / cerebral ischemia (reviewed by Graham et al. Comp Med. 2004 54 (5): 486-96), such as the MCAO rat model. In this case, stenosis due to ligation surgically applied around the cerebral artery mimics the action of cerebral infarction by restricting blood flow to the brain and causing ischemia and subsequent neurodegeneration. Yes.

[102] In a transient MCAO model, a candidate population of bone marrow cells selected in the OGD assay is administered by constant instillation into the blood flow of transient MCAO rats. One skilled in the art can determine an appropriate dosage, which may range, for example, from 7.5 × 10 ⁴ cells to 3.75 × 10 ⁷ cells. The cells can be injected into either the jugular vein (IV) or the carotid artery (ICA), for example. Controls consist of administration of an equal volume of cryopreservation medium or saline. The cells contained in the candidate bone marrow cell population then migrate to the infarct site caused by transient MCAO.

  [103] Neurological function in the presence or absence of a population of OGD selected bone marrow cells is then assessed by a modified Bederson Neurologic Test at various time points after infarction. Rats are then sacrificed and infarct volume and host cell viability are measured by hematoxylin and eosin (H & E) or Nissl staining of brain tissue sections from treated and untreated MCAO rats. A population of bone marrow cells is then selected according to their ability to improve neurological function, increase host cell viability and reduce infarct volume by 28 days after MCAO compared to control animals.

  Combination therapy to treat neurodegeneration caused by cerebral infarction

  [104] Mimics permanent arterial occlusion with clots by stopping blood flow through the middle cerebral artery for an extended period of time. Transient occlusion that restores and reperfuses the blood flow through the cerebral artery for a limited period of time, and then enters the stroke penumbra by thrombolysis or mechanical clot removal immediately after the arterial occlusion caused by cerebral infarction. It shall mimic a therapy that restores blood flow.

  [105] In many respects, administration of NCS-01 cell populations to transient MCAO rats is a consequence of thrombolysis or mechanical clot removal including angioplasty or surgical stent implantation. Mimic reperfusion of an occluded artery.

  [106] When neurodegeneration occurs due to cerebral infarction, the NCS-01 cell population can be used in combination with thrombolytic therapy for the treatment of neurodegeneration caused by ischemia. Descriptions of thrombolytic agents and their administration are found, for example, in U.S. Patent Nos. 5,945,432 and 6,821,985.

  [107] Thrombolytic agents that are injected after an ischemic event can be administered before, together with, or after injection of the NCS-01 cell population.

  [108] Examples of mechanical measures for improving blood flow to the stroke penumbra that can be used in combination with the infusion of the disclosed NCS-01 cell composition include, but are not limited to, angioplasty or surgical techniques well known in the art. Stent implantation is included.

  [109] The invention is described in further detail with reference to the following examples.

  [110] The examples demonstrate how to isolate, select and use a subpopulation of bone marrow cells to treat neurodegeneration in accordance with the present invention. It is understood that the method steps described in these examples are not meant to be limiting. Other objects and advantages of the invention other than those set forth above will become apparent from the examples; the examples are not intended to limit the scope of the invention.

Example 1: Separation of NCS-01 cell population 1) Separation of candidate bone marrow cell population
[111] A heterogeneous population of bone marrow cells was isolated by the following preparation method:
Human untreated bone marrow was collected by a qualified vendor from a pre-screened healthy 50-year-old donor. Bone marrow was taken from a donor that was not pretreated with a cytostatic agent such as 5-fluorouracil;
Treated or untreated bone marrow is then seeded onto a tissue / cell culture plastic surface at low density (10 ² to 10 ⁶ cells / cm ² ) and cultured in the presence of serum-containing medium;
After several days of cell attachment to plastic, unattached cells were removed by washing; and • The adherent cell population was cultured to near confluence in serum-containing medium; Continuous passage was performed at about 7 consecutive passages or less, and cultured cells were seeded at low density at each passage.

  [112] In order to select the optimal culture conditions for isolating a population of bone marrow cells capable of treating neurodegeneration, the cell population is first selected as cell density at seeding, cell passage number, culture medium composition, or The cells were grown under various culture conditions such as cell fractionation (see Table I).

  [113] Alpha-MEM supplemented with 2 mM GlutaMax (Invitrogen) and 10% fetal calf serum (FBS, HyClone or GIBCO) from untreated bone marrow, or after density fractionation or ACK lysis, or Tissue / cell culture plastics were seeded in the presence of α-MEM (Mediatech) supplemented with 2 mM GlutaMax (Invitrogen) and 10% fetal calf serum (FBS, HyClone or GIBCO), or serum-free medium (StemPro) . After washing away unattached cells, the attached cells were grown to near confluence. Cells were then passaged for a total of 3, 4, 5 or 6 passages.

[114] Candidate bone marrow cell populations were then tested in their in vitro OGD assay and in vivo MCAO test for their ability to treat neurodegeneration.

2) Primary screening using in vitro oxygen / glucose depletion (OGD) protocol
[115] Various parameters in the preparation process outlined above, such as bone marrow preparation in the presence or absence of density fractionation, seeding density, passage number, culture medium, and / or combinations thereof, An optimal method was determined to isolate candidates for a population of bone marrow cells that could be assessed by an experimental / glucose depletion (OGD) experimental protocol.

  [116] The in vitro OGD model was chosen as the primary screen because it mimics neurodegeneration caused by cerebral infarction. Specifically, whether a particular sub-population of bone marrow cells can prevent the death of neural cells in culture and induce the secretion of neuroprotective trophic factors such as bFGF and IL-6, Tested by OGD.

  [117] In an in vitro oxygen glucose depletion (OGD) model, primary mixed cultures of rat neurons and astrocytes (in a 1: 1 ratio) have OGD injury (8% oxygen; glucose-free Earle) (Salt solution) was applied for 90 minutes, returned to physiological conditions, and allowed to stand for 2 hours. The OGD-treated neuron-astrocyte co-culture was added with a candidate for the bone marrow cell population, and further kept for 3 hours. Viability of neural cells using standard trypan blue staining and MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) method immediately after OGD and 5 hours after OGD Evaluated.

Cell culture
[118] Primary mixed cultures of rat neurons and astrocytes were maintained by culture according to the vendor protocol (CAMBREX, MD). Immediately after thawing, cells (4 × 10 ⁴ cells / well) were seeded in polylysine-coated 96-well plates and Neurobasal media (neural basal media) (GIBCO, Calif.) [2 mM L-glutamine, 2% Of B27 (GIBCO, CA) and 50 U / ml penicillin and streptomycin] at 37 ° C. in a humidified atmosphere containing 5% CO ₂ for 7-10 days. The purity of neuronal and astrocyte cell populations was then assessed by MAP2 and GFAP immunostaining, respectively, and was found to be greater than 99%.

Co-culture with oxygen glucose depletion (OGD) and bone marrow cell population candidates
[119] OGD injury model conditions were given to cultured cells according to a slight modification of the previous description (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 MgSO ₄ , 1 mM NaH. ₂ PO ₄ , 26.2 mM NaHCO ₃ , 0.01 mM glycine, 1.8 mM CaCl ₂ , pH adjusted to 7.4. Cultured cells were placed in a humidified chamber and equilibrated for 15 minutes with a continuous flow of 92% N ₂ and 8% O ₂ gas. After reaching equilibrium, the chamber was sealed and placed in a 37 ° C. incubator for 90 minutes. After this period, OGD was terminated by adding glucose to the culture medium and returning the culture to a standard 95% O ₂ and 5% CO ₂ incubator. A 2 hour 'reperfusion' was then performed in standard medium and normoxic conditions, after which a candidate bone marrow (BM) cell population was added to the OGD-treated mixed neuron-glia culture for about 3 hours. The supernatant and bone marrow cell population was then separated from the mixed culture by washing. The cells were then subjected to cell viability and immunocytochemistry tests according to the following, and the amount of secreted trophic factors was measured by a commercially available ELISA assay.

Cell viability assay
[120] Cell viability was assessed at two time points: immediately after OGD and 5 hours after OGD (ie, 2 hours of reperfusion + 3 hours of treatment with a selected population of bone marrow cells). For post-OGD viability assays, the supernatant containing bone marrow-derived cells was separated from the attached mixed neural cell culture. The trypan blue staining method was performed, and the average viable cell count was calculated in three randomly selected regions (0.2 mm ² ) in each well (n = 5 per treatment condition), and the cell viability for each treatment condition was calculated. I investigated. In addition, trypan blue staining was performed on a subset of bone marrow derived cells harvested as a pellet from the supernatant.

ELISA assay
[121] It is presumed that trophic factors secreted by bone marrow-derived cells, such as bFGF and IL-6, and potential neurotrophic factors are involved in the treatment of neurodegeneration mimicked by OGD culture conditions. Thus, measuring the amount of these molecules secreted into the culture medium provides a basis for evaluating a candidate population of bone marrow cells that can treat neurodegeneration in vivo. Supernatants are collected from candidates for neural and bone marrow cell populations co-cultured under standard culture conditions or exposed to OGD, and the presence of trophic factor secretion is determined using a commercially available ELISA kit according to the manufacturer's instructions. analyzed.

  [122] The results of OGD analysis of bone marrow-derived cell populations treated according to the parameters shown in Table I above are shown in FIGS. 1A and 1B.

  [123] Based on the results shown in FIGS. 1A and 1B, αMEM + 10% FBS was selected as the optimal cell culture medium, and untreated bone marrow was treated by density fractionation (eg, Ficoll-Paque or Percoll) or ACK lysis It was found to be superior to bone marrow. It was found that the optimal passage number for untreated bone marrow cells in αMEM + 10% FBS medium was 7 passages or less.

3) Secondary screening and selection of candidate bone marrow cell populations using an in vivo middle cerebral artery occlusion rat model
[124] Based on the results obtained in the primary screening in an in vitro OGD model, the neuronal deficits and infarct volumes of MCAO rats treated with a candidate population of bone marrow cells were compared to MCAO rats treated with saline alone. In doing so, each candidate bone marrow cell population was evaluated for biological activity in treating neurodegeneration.

Middle cerebral artery occlusion (MCAO) surgery
[125] Animals were anesthetized with isoflurane (1.5% -2.5%, containing oxygen). The scalp was shaved and wiped with alcohol and chlorhexidine surgical scrub. The animal was then placed in a stereotaxic device. A midline sagittal incision approximately 2.5 cm long was made, starting slightly behind the eye, and the skull area was exposed using the rounded edge of a spatula. Baseline (ie, before surgery for stroke) laser Doppler recordings were determined from the following coordinates (AP: +2.0, ML: ± 2.0) with Bregma as the reference point. The skin on the ventral side of the neck was shaved from the jaw to the manubrium and wiped with alcohol and chlorhexidine surgical scrub. The animals were then moved under a surgical microscope. A skin incision was made in the upper right carotid artery. The external carotid artery was isolated and ligated as distally as possible. The occipital artery was cauterized. In some cases there were one or two branches extending from the external carotid artery, which also needed to be cauterized. A second ligature was placed proximal to the external carotid artery and cut between the ligatures. The pterygopalatine artery was ligated. This was followed by provisional sutures around the common carotid artery to provide tension and restrict blood flow. Using ligation, the proximal stump of the external carotid artery was pulled back to efficiently extend the carotid bifurcation. An incision was made at the stump of the external carotid artery using a micro scissor, and a 4-0 nylon thread pre-processed at the end was inserted and passed through the internal carotid artery until resistance was felt (about 15-17 mm). This effectively blocks the middle cerebral artery (MCA). The thread was secured in place with a ligature around the proximal stump of the external carotid artery. The contralateral common carotid artery was isolated and fixed with a temporary ligation. The skin incision was closed with staples. The animals were then fixed to a stereotaxic device for laser Doppler recording, confirming successful MCA occlusion. After 5 minutes, the contralateral common carotid artery ligation was removed. Isoflurane was stopped and the animals were placed in a recovery cage and placed on a warming blanket. After 60 minutes, the animals were again anesthetized with isoflurane and the incision opened for testing in this transient model. The thread causing the occlusion was removed and the stump of the external carotid artery was ligated near the carotid bifurcation. The skin incision was closed with staples. The animals were again fixed to the stereotaxic device for laser Doppler recording, confirming successful reperfusion. Finally, the animals were placed in a recovery cage and placed on a thermal blanket.

Nerve function test
[126] A well-recognized modified Vaderson neurological test was performed on each rat; this involved determining a score from each of the following:
• Pulling the contralateral hind paw; this measures the ability of the animal to undo it after shifting the hind paw outward 2-3 cm and is rated from 0 (immediate pull back) to 3 (do not return);
-Beam walking ability; rated from 0 (rat that immediately crosses a beam of width 2.4-cm, length 80-cm) to 3 (rat that cannot stay on the beam for 10 seconds); and It measures the ability to cling to a 2-mm diameter steel rod and is rated from 0 (rats with normal forelimb gripping behavior) to 3 (rats that cannot be gripped with forelimbs).

  [127] Scores from all three trials were evaluated over approximately 15 minutes on each evaluation day. An average score was calculated from the three trials to determine a total nerve defect score ranging from 0 (normal nerve function) to a maximum of 3 (serious nerve defect). Therefore, the higher the score, the greater the neurological deficit.

  [128] Based on pilot studies, a score above about 2.5 is an indication that an animal has a neurological deficit characteristic of stroke.

Tissue findings Brain slice specimen
[129] Design brain slices to identify areas of brain injury. Rats were euthanized on day 7 or 28 after MCA occlusion and perfused with transcardiac perfusion of saline followed by 4% paraformaldehyde. The brain was then fixed in 4% paraformaldehyde and then immersed in 25% sucrose. Each forebrain was cut into 30 μm thick coronal tissue sections with anterior-posterior coordinates corresponding to Bregma 5.2 mm to Bregma-8.8 mm for each animal.

Measurement of infarct volume
[130] At least four coronary tissue sections per brain were processed for hematoxylin and eosin (H & E) staining or Nissl staining. In order to confirm cerebral infarction, an indirect lesion area obtained by subtracting the intact area of the contralateral hemisphere from the area of the contralateral hemisphere was used.

  [131] Lesion volume was presented as the percent volume of the lesion compared to the contralateral hemisphere. Histological measurements of lesion volume were performed using hematoxylin and eosin (H & E) staining or Nissl staining and representative images were acquired digitally and processed by NIH Image J software and quantitative image analysis. The lesion volume was determined according to the following formula:

  [132] Section thickness x sum of infarct area in whole brain section

  [133] In order to minimize the artifacts caused by postischemic edema of the infarct area, the infarct of the ipsilateral hemisphere is subtracted from the non-infarct area of the ipsilateral hemisphere from the total intact area of the contralateral hemisphere. The area was measured indirectly.

Measurement of cell viability in the peri-infarct region due to ischemia
[134] Surviving cells in this ischemic area were counted using a randomly selected high power field corresponding to the peri-infarct area of the cortex (Yasuhara et al., Stem Cells and Dev, 2009). To estimate the viability of host nervous system cells within the ischemic cortex area, Nistle staining was performed using crystal violet solution (Sigma, St. Louis, MO) and randomly selected cortical areas in 3 sections and corresponding The field of contralateral intact cortex was photographed (Carl Zeiss, Axiophoto ² ) and the number of cells was determined by counting cells in a randomly selected high power field (28,800 μm ² ). The percentage of conserved neurons in the damaged cortex to the intact side was calculated and used for statistical analysis. Brain sections were blind coded and the total number of stained cells counted was corrected by the Abercrombie equation.

Screening candidate bone marrow cell populations using the MCAO stroke animal model
[135] Three (IV-administered) or six (ICA-administered) male rats / group received 1 hour of transient MCAO followed by bone marrow derived cells (NCS-01) isolated according to saline or the protocol described above. 1 ml of injection medium containing 7.5 × 10 ⁶ cells (referred to as cell population) was injected. Animals were followed up to 7 days after cell administration.

  [136] FIGS. 1C and 1D show that NCS-01 bone marrow cell populations administered IV or ICA provided substantial neurological and pathological benefits when administered to rats with transient MCAO Indicates. Furthermore, NCS-01 prevented the death of host cells by treating neurodegeneration induced by ischemia, resulting in reduced infarct volume and improved nerve deficits.

  [137] The primary in vitro and secondary in vivo screens were repeated until the optimal sub-population of bone marrow-derived cells (referred to as NCS-01 cell population) capable of treating neurodegeneration was reliably and reproducibly generated by the method. It was.

  [138] This optimized NCS-01 cell population was retested in an in vitro OGD model to confirm anti-neurodegenerative activity in coculture of human neurons and astrocytes (see Figure IE) and in vivo rat Retested with the MCAO model (FIG. 1F). The experiment shown in FIG. 1F also shows that the ability of the NCS-01 cell population to treat neurodegeneration is dose dependent.

Example 2: Standardized preparation method for preparing NCS-01 cell population capable of treating neurodegeneration
[139] Untreated whole bone marrow is taken from a mammal that has not been pretreated with any cytostatic or antimetabolite, such as 5-fluorouracil (5-FU). Since this bone marrow has not been processed by density fractionation or ACK lysis, the starting whole bone marrow cell population may contain both hematopoietic and non-hematopoietic cells, as well as nucleated and non-nucleated bone marrow cells. is there.

[140] The untreated bone marrow is diluted and seeded at low density (10 ⁵ to 10 ⁶ cells / cm ² ) on a tissue / cell culture plastic surface, serum-containing medium (2 mM GlutaMax (Invitrogen) and 10% Α-MEM supplemented with fetal calf serum (FBS, HyClone or GIBCO) or alpha-MEM supplemented with 2 mM GlutaMax (Invitrogen) and 10% fetal calf serum (FBS, HyClone or GIBCO) (Mediatech)). The cell culture is then incubated for 72 hours at 37 ° C., 5% CO ₂ , 80% RH (relative humidity).

[141] Rinse cells with D-PBS to remove non-adherent cells and RBC from cell culture plastic, followed by complete media exchange with supplemented α-MEM, which is used for all subsequent feeds. Cell cultures (passage 0 or p0) were then incubated at 37 ° C., 5% CO ₂ , 80% RH.

[142] For passage 1, the cells are rinsed with D-PBS and the cells attached to the plastic are detached with a cell dissociation reagent. Dissociated cells are harvested by centrifugation at 300 g (about 1000 rpm) for 8-10 minutes. The pelleted cells are resuspended in supplemented α-MEM and seeded on a tissue / cell culture plastic surface at a density of about 750 cells / cm ² .

  [143] Cells are cultured to near confluence and then passaged.

[144] For passage 2, cells are harvested as described above and again seeded onto the tissue / cell culture plastic surface at a density of about 750 cells / cm ² .

  [145] For passage 3, cells are harvested using a cell dissociation reagent and centrifuged as described above. The pelleted cells are then resuspended and pooled. Cells are resuspended in cryopreservation medium and 1 mL of cell suspension is subdivided into Cryovial (Nunc). Vials are frozen in a controlled speed freezer and, once the freezing program is complete, are transferred onto dry ice for permanent storage in a gas phase liquid nitrogen freezer.

  [146] A vial containing cells from passage 3 (p3) becomes the MCB.

[147] One MCB vial was thawed and the recovered cells (passage 4 or P4) were tissued at a density of approximately 750 cells / cm ^{2 in} α-MEM supplemented with 10% FBS and GlutaMAX ™. / Seed in cell culture plastic. The cells are then incubated at 37 ° C., 5% CO ₂ , 80% RH.

  [148] For passage 4, the cells are cultured to near confluence and then further passaged. Cells are harvested as described above and seeded onto new tissue / cell culture plastic surfaces.

  [149] For passage 5, harvest and freeze cells according to the method described above for MCB. Passage 5 (p5) cells subdivided into vials and cryopreserved become WCB.

[150] If necessary, one of the MCB vials was thawed and the harvested cells were collected at a density of about 750 cells / cm ^{2 in} α-MEM supplemented with 10% FBS and GlutaMAX ™. Seed in cell culture plastic. The cells are then incubated at 37 ° C., 5% CO ₂ , 80% RH.

  [151] For passage 6, the cells are cultured to near confluence and then further passaged. Cells are harvested as described above and seeded onto new tissue / cell culture plastic surfaces.

  [152] The medium is changed between passages (from WCB to passage 6, and passage 6 and passage 7).

  [153] For passage 7, harvest and freeze cells according to the method described above for MCB.

  Example 3: Treatment with NCS-01 cells of neurodegeneration caused by permanent middle cerebral artery occlusion (MCAO) compared to transient ones

  [154] The infarct volume and nerve function of rats with NCS-01 treated permanent MCAO were compared to those of rats with NCS-01 treated transient (60 min) MCAO.

  [155] Transient MCAO mimics the treatment of an arterial occlusion caused by a stroke by current standard clinical measures to restore blood flow to the stroke penumbra. These measures include the administration of thrombolytic agents and measures such as angioplasty and / or vascular stent insertion with mechanical removal of blood clots.

[156] Groups of 3 or 6 rats received either permanent MCAO or transient MCAO (with reperfusion). Either 1 ml saline or 7.5 × 10 ⁶ NCS-01 cells were then administered ICA 24 hours after ischemia and rats were monitored until day 28. Neurological function was then assessed by a modified Vaderson neurological test. The transient occlusion model mimics the situation where a stroke patient is treated with tPA or a clot removal procedure.

  [157] The results of FIG. 2 show significant histological benefit (infarction volume; panel A) and clinical benefit (modified Vaderson scale; panel B) for treatment with the NCS-01 cell population in both MCAO paradigms. Indicates. The benefit was 2-3 times greater in the transient occlusion model than in the permanent occlusion model. The time course of the neural response (Panel B) showed a steady improvement until day 28 after infarction, with an average of 11% for non-reperfusion (permanent occlusion) and untreated controls over this period from reperfusion. (Transient occlusion) and 67% for NCS-01 treated animals.

  [158] Unexpectedly, NCS-01 was more effective in the treatment of symptoms in the transient occlusion model; this combined with NCS-01 in combination with clot removal, It suggests that maximum efficacy may be obtained when applied after any of the clot removal with mechanical instruments.

Example 4: Comparison of NCS-01 cells with other bone marrow derived cells 1) Separation of a population of bone marrow cells according to Li et al.
[159] A population of bone marrow cells was prepared in strict accordance with the description of the publication Li et al. Journal of Cerebral Blood Flow and Metabolism (2000) 20: 131 1-1319 (hereinafter referred to as the Li bone marrow cell population).

[160] Primary cultured bone marrow cells were collected from adult mice administered intraperitoneally with the antimetabolite 5-fluorouracil (5-FU 150 mg / kg) two days before collection (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 dissociated until it became a milky homogeneous single cell suspension. Red blood cells were removed from the bone marrow using 0.84% NH ₄ Cl and the number of nucleated bone marrow cells was measured using a hemocytometer. 2 × 10 ⁶ nucleated bone marrow cells were seeded in Iscove's Modified Dulbecco's medium supplemented with fetal calf serum (10%) in a tissue culture flask. After 3 days of incubation, the cells adhered tightly to the plastic and they were resuspended in fresh Iscove's modified Dulbecco medium in a new flask and grown for 3 additional passages.

  2) Comparison of Li and NCS-01 cell populations in in vitro OGD assay [161] Two lots of NCS-01 prepared from the same MCB and different WCBs of bone marrow, together with the Li cell population, are outlined in FIG. Were tested in an in vitro OGD model.

  [162] As shown in FIG. 4, both lots of NCS-01 cells produced the same increase in secretion of both IL-6 and bFGF. Thus, the NCS-01 cell population obtained by the optimized preparation method described above reliably treated neurodegeneration in an in vitro OGD assay.

  [163] In contrast, Li cell populations isolated strictly according to the description in the Li (2000) publication produced 4-5 times less bFGF and IL-6 than NCS-01 cell populations in the OGD assay.

  2) Comparison of Li cell population and NCS-01 cell population in in vivo MCAO assay

  [164] The ability of NCS-01 and Li cell populations to treat neurodegeneration in vivo was tested in vivo in the MCAO rat model. The effects of these cells on host cell viability, infarct volume, and nerve defects are shown in FIGS. 5A and 5B. Consistent with the previous study, the NCS-01 cell population prevented host cell death by treating neurodegeneration induced by ischemia (see FIG. 5A), resulting in reduced infarct volume. And improved neurological deficits (see FIG. 5B).

  [165] In contrast, the Li cell population did not show any statistically significant activity on infarct volume or nerve function. Therefore, these data indicate that the ability of the NCS-01 population to treat neurodegeneration depends on the preparation method and that the NCS-01 cell population is distinct from the cell population described in the Li 2000 publication. Prove it.

  [166] Any patents, patent applications, publications, or other disclosures mentioned herein are hereby incorporated by reference in their entirety. Anything mentioned or incorporated in this specification that is inconsistent with an existing definition, description, or other disclosure set forth herein is hereby incorporated by reference. It is used only to the extent that no contradiction arises between them.

Claims (34)

A therapeutic agent for neurodegeneration due to ischemia , including a heterogeneous subpopulation of bone marrow cells ,
A therapeutic agent comprising a heterogeneous bone marrow cell subpopulation that is injected into the bloodstream of a mammal with neurodegeneration due to ischemia and reduces nerve defects caused by the neurodegeneration due to ischemia,
The heterogeneous sub-population of bone marrow cells is
a) collecting a population of bone marrow cells from human untreated bone marrow;
b) seeding the population of bone marrow cells on a plastic surface at a low density of 10 ⁵ cells / cm ² to 10 ⁶ cells / cm ² ;
c) washing the seeded cell population to remove non-adherent cells;
d) culturing adherent cells from said washed cell population to confluence in serum-containing medium;
e) The cultured cells are continuously passaged in serum-containing medium at 7 consecutive passages or less, and at each passage, the cultured cells are seeded at a low density of about 750 cells / cm ² ;
f) prepared by a method comprising collecting said heterogeneous bone marrow cell subpopulation;
A therapeutic agent for neurodegeneration caused by ischemia . The therapeutic agent for neurodegeneration due to ischemia according to claim 1, wherein the neurodegeneration is caused by cerebral infarction. In combination with additional treatments to increase blood flow through the occluded blood vessels,
Infusion of the therapeutic agent in combination with the additional treatment significantly reduces neurological deficits after either infusion of the subpopulation of bone marrow cells with or without additional treatment alone.
The therapeutic agent for neurodegeneration caused by ischemia according to claim 2. The therapeutic agent for neurodegeneration due to ischemia according to claim 3, wherein the additional treatment for increasing the blood flow through the occluded blood vessel is performed simultaneously with the injection of the therapeutic agent . The therapeutic agent for neurodegeneration due to ischemia according to claim 3, wherein an additional treatment for increasing blood flow through the occluded blood vessel is performed before the injection of the therapeutic agent . The therapeutic agent for neurodegeneration due to ischemia according to claim 3, wherein the occluded blood vessel is occluded by a blood clot. The therapeutic agent for neurodegeneration due to ischemia according to claim 6, wherein the clot in the occluded blood vessel is due to rupture of atherosclerotic plaque. The therapeutic agent for neurodegeneration due to ischemia according to claim 3, wherein the additional treatment for increasing blood flow through the occluded blood vessel comprises administration of a thrombolytic agent . The therapeutic agent for neurodegeneration due to ischemia according to claim 8, wherein the thrombolytic agent is at least one selected from the group consisting of streptokinase, urokinase, and recombinant tissue plasminogen activator. The therapeutic agent for neurodegeneration due to ischemia according to claim 9, wherein the recombinant tissue plasminogen activator is alteplase, reteplase, desmoteplase or tenecteplase. The therapeutic agent for neurodegeneration due to ischemia according to claim 3, wherein the additional treatment for increasing blood flow through the occluded blood vessel includes mechanically removing a blood clot from the occluded blood vessel. The therapeutic agent for neurodegeneration due to ischemia according to claim 3, wherein the additional treatment for increasing blood flow through the occluded blood vessel further includes capturing a blood clot with a filter. 4. The therapeutic agent for neurodegeneration due to ischemia according to claim 3, wherein the additional treatment for increasing blood flow through the occluded blood vessel comprises performing angioplasty and / or vascular stenting. The therapeutic agent for neurodegeneration due to ischemia according to claim 1, which is injected into a vein or artery. The therapeutic agent for neurodegeneration due to ischemia according to claim 1, which is injected into the carotid artery. The therapeutic agent for neurodegeneration due to ischemia according to claim 1, which is injected into the brain. A therapeutic agent for neurodegeneration due to ischemia , including a heterogeneous subpopulation of bone marrow cells ,
A therapeutic agent comprising a heterogeneous bone marrow cell subpopulation that is injected into the bloodstream of a mammal with neurodegeneration caused by ischemia and reduces the volume of infarct caused by neurodegeneration due to ischemia,
The heterogeneous sub-population of bone marrow cells is
a) collecting a population of bone marrow cells from human untreated bone marrow;
b) seeding the population of bone marrow cells on a plastic surface at a low density of 10 ⁵ cells / cm ² to 10 ⁶ cells / cm ² ;
c) washing the seeded cell population to remove non-adherent cells;
d) culturing adherent cells from said washed cell population to confluence in serum-containing medium;
e) The cultured cells are continuously passaged in serum-containing medium at 7 consecutive passages or less, and at each passage, the cultured cells are seeded at a low density of about 750 cells / cm ² ;
f) prepared by a method comprising collecting said heterogeneous bone marrow cell subpopulation;
A therapeutic agent for neurodegeneration caused by ischemia . The therapeutic agent for neurodegeneration caused by ischemia according to claim 17, wherein the neurodegeneration is caused by cerebral infarction. In combination with additional treatments to increase blood flow through the occluded blood vessels,
Infusion of the therapeutic agent in combination with the additional treatment significantly reduces the volume of the infarct after either the additional treatment alone or the injection of a subpopulation of bone marrow cells without additional treatment.
The therapeutic agent for neurodegeneration caused by ischemia according to claim 17. 20. The therapeutic agent for neurodegeneration due to ischemia according to claim 19, wherein the additional treatment for increasing blood flow through the occluded blood vessel comprises administration of a thrombolytic agent . 21. The therapeutic agent for neurodegeneration due to ischemia according to claim 20, wherein the thrombolytic agent is at least one selected from the group consisting of streptokinase, urokinase, and recombinant plasminogen activator. The therapeutic agent for neurodegeneration due to ischemia according to claim 21, wherein the recombinant plasminogen activator is alteplase, reteplase, desmoteplase or tenecteplase. 20. The therapeutic agent for neurodegeneration due to ischemia according to claim 19, wherein the additional procedure for increasing blood flow through the occluded blood vessel comprises performing angioplasty and / or stenting. 20. The therapeutic agent for neurodegeneration due to ischemia according to claim 19, wherein the additional treatment for increasing blood flow through the occluded blood vessel includes mechanical removal of a blood clot near the infarct site. The therapeutic agent for neurodegeneration due to ischemia according to claim 17, which is injected into a vein or artery. The therapeutic agent for neurodegeneration due to ischemia according to claim 17, which is injected into the carotid artery. The therapeutic agent for neurodegeneration due to ischemia according to claim 17, which is injected into the brain. A treatment kit for neurodegeneration due to ischemia comprising a thrombolytic agent and a heterogeneous bone marrow cell subpopulation comprising:
The heterogeneous sub-population of bone marrow cells is
a) collecting a population of bone marrow cells from human untreated bone marrow;
b) seeding the population of bone marrow cells on a plastic surface at a low density of 10 ⁵ cells / cm ² to 10 ⁶ cells / cm ² ;
c) washing the seeded cell population to remove non-adherent cells;
d) culturing adherent cells from said washed cell population to confluence in serum-containing medium;
e) The cultured cells are continuously passaged in serum-containing medium at 7 consecutive passages or less, and at each passage, the cultured cells are seeded at a low density of about 750 cells / cm ² ;
f) prepared by a method comprising collecting said heterogeneous bone marrow cell subpopulation;
Treatment kit for neurodegeneration caused by ischemia . The treatment kit for neurodegeneration due to ischemia according to claim 28, wherein the thrombolytic agent is at least one selected from the group consisting of streptokinase, urokinase, and recombinant tissue plasminogen activator. 30. The treatment kit for neurodegeneration due to ischemia according to claim 29, wherein the recombinant tissue plasminogen activator is alteplase, reteplase, or tenecteplase. A kit for the treatment of neurodegeneration due to ischemia , comprising a heterogeneous subpopulation of bone marrow cells and means for mechanical removal of blood clots,
The heterogeneous sub-population of bone marrow cells is
a) collecting a population of bone marrow cells from human untreated bone marrow;
b) seeding the population of bone marrow cells on a plastic surface at a low density of 10 ⁵ cells / cm ² to 10 ⁶ cells / cm ² ;
c) washing the seeded cell population to remove non-adherent cells;
d) culturing adherent cells from said washed cell population to confluence in serum-containing medium;
e) The cultured cells are continuously passaged in serum-containing medium at 7 consecutive passages or less, and at each passage, the cultured cells are seeded at a low density of about 750 cells / cm ² ;
f) prepared by a method comprising collecting said heterogeneous bone marrow cell subpopulation;
Treatment kit for neurodegeneration caused by ischemia . A composition for treating neurodegeneration due to cerebral infarction for treating neurodegeneration caused by cerebral infarction , comprising a thrombolytic agent, a heterogeneous bone marrow cell subpopulation, and a carrier for injecting the composition A composition comprising:
The heterogeneous sub-population of bone marrow cells is
a) collecting a population of bone marrow cells from human untreated bone marrow;
b) seeding the population of bone marrow cells on a plastic surface at a low density of 10 ⁵ cells / cm ² to 10 ⁶ cells / cm ² ;
c) washing the seeded cell population to remove non-adherent cells;
d) culturing adherent cells from said washed cell population to confluence in serum-containing medium;
e) The cultured cells are continuously passaged in serum-containing medium at 7 consecutive passages or less, and at each passage, the cultured cells are seeded at a low density of about 750 cells / cm ² ;
f) prepared by a method comprising collecting said heterogeneous bone marrow cell subpopulation;
A composition for the treatment of neurodegeneration due to cerebral infarction . The composition for treating neurodegeneration due to cerebral infarction according to claim 32, wherein the thrombolytic agent is at least one selected from the group consisting of streptokinase, urokinase, and recombinant tissue plasminogen activator. . 34. The composition for treating neurodegeneration due to cerebral infarction according to claim 33, wherein the recombinant tissue plasminogen activator is alteplase, reteplase, or tenecteplase.