Claims:1. A method of preparation of a therapeutic composition including mesenchymal stem cells derived from cord tissues and dental pulp, capable of releasing both anti-inflammatory and neuronal specific growth factors, for treating ischemic brain stroke wherein the composition can be administered into patient intra-thecally comprising the steps of:
(a) Culturing and expanding the above mesenchymal tissue fragments in a growth medium under suitable conditions and sufficient time in a mother plate;
(b) Transferring the cultured adherent explants from mother plate to a fresh coated tissue culture plate ensuring that the stromal side is in contact with the culture dish;
(c) Trypsinizing the attached cells from the adherent plate surface and re-seeding for therapeutic dosage in the fresh coated tissue plate;
(d) Supplementing the cells obtained in step (c) with hypoxia for 3 days;
(e) Resuspending the cells obtained in step (d) in injectable nerve growth factor (NGF) for infusion; and
(f) Cryopreserving the obtained composition before injecting into the patient.
, Description:REFERENCE TO RELATED APPLICATIONS

This application claims priority to Indian Patent Application No. 932/CHE/2013 filed on March 04, 2013 entitled “Novel method of progenitor cell expansion”, which is hereby expressly incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of clinical application of stem cell technology and more particularly to a novel cell based brain stroke therapy relevant composition with characteristics that triggers engraftment and homing upon infusion.

BACKGROUND OF THE INVENTION

Stroke is a very common cause of accidental disability in the developed and developing world. It is to be noted that stroke affects one in every six people worldwide, and is the leading cause of adult disability. With an aging population, the incidence and prevalence of stroke are predicted to rise. Stroke is an acute-onset clinical syndrome that develops following a vascular insult to the brain. Brain ischemia resulting from thromboembolism or less frequently, in situ thrombosis, constitutes 80-85% and haemorrhage resulting from hypertension or vessel wall pathology constitutes 15-20% of all strokes. Following vascular occlusion, a complex chain of events occurs at a molecular level, leading to irreversible tissue injury, including failure of energy synthesis, loss of trans-membrane ionic gradients dependent on active transport, cell depolarization, and excitotoxicity due to the excess release of excitatory neurotransmitters. In the region with severely reduced blood flow (the ischemic core), these processes result in rapid cell necrosis affecting all the cellular elements (neurons, glia, and blood vessels). A region around the core (the ischemic penumbra) transiently maintains a collateral blood supply sufficient for cell viability. Restoring perfusion can salvage penumbral tissue and timely recanalization is the most robust predictor of good clinical prognosis following ischemic stroke. Early thrombolysis with intravenous recombinant tissue plasminogen activator increases the likelihood of recanalization and a recovery to independence defined on scales of disability and handicap. Alternative reperfusion strategies have not yet shown benefit. Secondary processes following ischemic injury and cell necrosis include an inflammatory response, with the activation of microglia, infiltration of tissue by neutrophils and macrophages from the blood and blood-brain barrier breakdown. Inflammatory mediators can act as chemo-attractants for both the endogenous and exogenous cells involved in tissue repair. At the network level, regions of the brain that were previously connected to the infarcted area reorganize, at least in terms of the brain activation patterns seen on functional magnetic resonance imaging (fMRI). Rehabilitation exploits the combination of functional reorganization and adaptation after stroke. Immediately after stroke, several events, including edema, deafferentation and inflammation occur around the infarct, and some early functional recovery can be attributed to the resolution of edema and inflammation. However, this is usually limited and other processes, including immunomodulation, angiogenesis, endogenous neurogenesis and altered gene expression, may be involved in the longer-term recovery of function.

The use of allogenic cells offer controller cell numbers and immediate availability, which may have advantages for acute treatment. Early clinical trials of both NSCs and MSCs are ongoing, and clinical safety data are emerging from limited numbers of selected patients. Ongoing research to identify prognostic imaging markers may help to improve patient selection, and the novel imaging techniques may identify biomarkers of recovery and the mechanism of action for cell therapies. The great majority of the early cell therapy clinical studies have involved adult-derived cells of either autologous or allogenic origin and no major safety issues have been identified till date, although the numbers of subject have been extremely small and follow-up periods limited. Several clinical trials are ongoing or planned, mostly using MSCsdelivered by IV infusion.

US 20100310570 entitled “Mapc treatment of brain injuries and diseases” discloses a method of treatment of various injuries, disorders, dysfunctions, diseases and the like of the brain with MAPCs, by administering to a subject suffering from such diseases such cells that (i) are not embryonic stem cells, or embryonic germ cells and not germ cells; (ii) can differentiate into atleast one cell type of each of atleast two of the endodermal, ectodermal and mesodermal embryonic lineages; (iii) with or without adjunctive immunosuppressive treatment.

US20130045189 entitled “Methods of treating stroke through administration of ctx0e03 cells” discloses methods to enhance the therapeutic effects of cellular or drug treatment in various diseases and disorders, particularly a method for treating a neurodegenerative disease in a patient or repairing neural damage caused by a disease or disorder, by administering a therapeutically effective amount of CTX0Eo3 cells to the patient, where the administration is performed intravenously or intra-arterially.

EP1949904 entitled “Cell therapy for chronic stroke” a method of treating stroke in a patient who has undergone a stroke, in which the method calls for administering at least 2 million suitable neuronal cells to at least one brain area involved in the stroke. Cells for administration in the above method are selected from the group consisting of hNT neuronal cells, HCN-1 cells, fetal cells, neural cells or a combination thereof.

However, the methods prescribed in the prior arts are not very efficient in treating both the induced inflammation and neuronal dysfunction of a stroke. The present invention hence comes up with a method of preparing a novel cell product to treat ischemic brain stroke comprising the cell products of cord tissue and tooth pulp derived mesenchymal stem cells (MSCs) employing a unique method.

SUMMARY OF THE INVENTION

The present invention discloses a method of preparation of a novel cell product to treat ischemic brain stroke comprising cord tissue derived MSCs which have been shown to be a great source of secretary trophic factors and tooth pulp derived MSCs which have superior neurogenic differentiation potential. The primary objective of the present invention is to prepare stem cell composition releasing both the anti-inflammatory and neuronal specific growth factors that is unique and optimal treatment tool for brain stroke patients. The stem cells are culture expanded and the product is cryopreserved before infusing into the patient's body.
The present invention discloses a method of preparing a therapeutic recipe with progenitor cell population grown in hypoxia which is clinically applied with injectable nerve growth factor routed intrathecally.

A method of preparation of a mesenchymal stem cell based product wherein the stem cells are derived from donated human umbilical cord, dental pulp, adipose and other stromal tissues comprising the steps of:

(a) Harvesting the above mesenchymal stem cells as per 932CHE2013;
(b) Trypsinizing the attached cells from the adherent plate surface and re-seeding for therapeutic dosage in passage 1;
(c) Supplementing passage 1 cells with hypoxia for 3 days; and
(d) Resuspending passage 1 cells in injectable nerve growth factor (NGF) for infusion

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the embodiments of the present invention, reference is now made to the following description taken in connection with the accompanying in which:

Figure 1 shows the microscopic view of cytometer locus with single cells scattered, as tested for mixture of different concentrations of cells as described in Example 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The clinical research conducted in this invention particularly concentrates on the MSCs derived from cord tissues and dental pulp. This method does not involve surgery or any in-vivo operational activities to collect the above mentioned tissues rather; they are collected form the discarded tissues or donated by a consented donor. The MSCs both from cord tissues and dental pulp, in equal proportion are isolated, cultured and expanded by the novel method as mentioned in the parent application 932/CHE/2013.

The MSCs from any of the cord and dental tissues are isolated and determined their relative capacities for sustained proliferation and multilineage differentiation. Individual tissue components are dissected, diced into atleast 1–2 mm3 fragments and aligned in explants cultures from which migrating cells were isolated using trypsinization.

The mesenchymal tissues isolated are then minced into fragments. The fragments are aligned and attached at regular intervals to coated dishes cultured in basal medium. Basal medium comprises of DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS; MSC-qualified Invitrogen) and penicillin–streptomycin–amphotericin B.

Cultures are maintained in a humidified atmosphere with 5% CO2 at 370C. Approximately 4 weeks after expansion, fibroblast-like adherent cells migrates from the tissue fragments. The adherent cells are allowed to reach confluency in the same coated tissue culture plate while the explants are transferred manually from mother plate to a fresh coated tissue culture plate using sterile forceps in the biosafety cabinet. The cells in the mother plate are trypsinized.

The removed tissue fragments, which are otherwise called as explants here are again aligned manually with the blunt sterile forceps so as to make sure that the stromal side is in contact with the surface of the tissue culture dish and cultured in the growth medium. Cultures are maintained in a humidified atmosphere supplementing them with hypoxia for 3 days.

Within the period of ten days, the adherent cells migrated from the tissue fragments. The cells are allowed to reach confluency in the same dish by culturing the cells with alternate day change of medium for one more week while the tissue fragments are manually transferred maximum thrice after, into fresh coated tissue culture dishes after the 12 day growth period in the mother plates.

The method allows the cells to adhere to the coated plastic petri dishes in spite of addition of further cell culture medium. Once the cells have adhered to the culture medium under gentle and constant temperature, the surface area for continued culturing is increased and also the final harvesting of cells is increased by the active detachment of the explants in the hypoxia conditions at 370C for 3 days. Further the obtained cells from passage 1 are re-suspended in injectable nerve growth factor for infusion.

The obtained product is cryo-preserved before administering into the patient’s body.

Example 1:

10-12 million cells trypsinized from passage 0 were supplemented with hypoxic conditions and the viability of passage 1 cells was evaluated for 8, 16, 48 and 72 hr using cell countess method. Table 1 depicts the percentage of cells viable at different time intervals in hypoxia condition.

Table 1

Time interval (in Hr) Percentage of viable cells
8 95
16 93
48 93
72 92

Example 2:

Different concentrations of passage 1(single cell suspension) as mentioned below were prepared at room temperature and the stability of the mix was evaluated by measuring the spheres or clusters formed after 1 hour of incubation.

(a) 1 million cells/1 ml of injectable NGF;
(b) 2 million cells/1 ml of injectable NGF;
(c) 5 million cells/1 ml of injectable NGF; and
(d) 10 million cells/1 ml of injectable NGF.

1 ul of each of the above mentioned cell concentration was mixed with trypan blue and examined for clusters of cells in hemocytometer. It has been reported that no clusters in 1-10 million cells/1ml of injectable NGF mixtures were found after one hour of room temperature incubation, supporting the hypothesis of no deleterious interference by NGF solvent in the product ready for infusion, as shown in Figure 1.