Claims:1. A system for predicting the cell differentiation capacity and functionality of any small molecule, comprises of:
(a) a diagnostic plate with multiple wells coated with active progenitor mesenchymal stem cells;
(b) a growth medium;
(c) plurality of differentiation medium;
(d) a positive control suitable for such differentiation medium used; and
(e) means for predicting the functionality of the test molecule.

2. The system as in Claim 1 wherein the quantity of the test molecule used shall be between 5-20mM in dimethyl sulphoxide (DMSO).

3. The system as in Claim 1 wherein it is used to predict the function of small molecule to regenerate/differentiate Ostecoytes, Chondrocytes, Adipocytes, Pancreatic beta cells, Cardiomyocytes , Epithelial cells etc from mesenchymal stem cells.

4. The system as in Claim 1 wherein the diagnostic plate comprises of 12 to 24 wells.

5. The system as in Claim 1 wherein the growth medium in the assay includes Dulbecco's Minimum Essential Medium (DMEM) and Epidermal Growth Factor (EGF).

6. The system as in Claim 1 wherein the differentiation medium can be selected from a group including Neurogenic Differentiation Medium (C28015) (Promega), Adipocyte Differentiation Medium (AL521) (Himedia), Epithelial Cell Differentiation Medium (ax0035) (Axol), DMSO: Dimethyl Sulfoxide, Osteocyte Differentiation Medium (AL522) (Himedia), Beta Cell Differentiation Medium,Cardio Myogenic Differentiation Medium (A25042SA) (Gibco) etc. depending on the functionality of the test molecule to be predicted.

7. The system as in Claim 6 wherein the positive control used for the differentiation medium listed will be selected from a group comprising Forskolin (Tocris) for Neurogenic Differentiation, Ascorbic acid (Lonza) for Adipogenic Differentiation, Valproic acid (Sigma) for Epithelial Cell Differentiation, Quercetin Hydrate (TCI chemicals) for Osteocyte Differentiation Medium, 5-Iodotubercidin (Sigma) for Beta Cell Differentiation Medium, and Phorbolmyristate acetate (Sigma) for Cardio Myogenic Differentiation.

8. An assay method for predicting the differentiation capacity and functionality of a molecule, comprising the steps of:
(a) Treating the diagnostic plate with test molecule (SM1; SM2) in sterile DMSO. SM1 is small molecule 1; SM2 is small molecule 2
(b) Supplementing the wells of the plate with 10mM of test molecule for every 72 hours;
(c) Monitoring the phenotype of the cells through the predicting means comparing with the positive controls used in the assay;
(d) Evaluating the genotype of the differentiated cells under the influence of the test molecule and comparing with the controls;
(e) Evaluating the toxicity of the test molecule on the human derived stem cell system; and
(f) Deriving the LD50 (Lethal Dose) of the test molecule on the human derived stem cell diagnostic system.
, 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 stem cell based product technology, particularly relates to a living human derived homogenous progenitor cell based kit that can predict the cellular differentiation and/or regenerating capacities of small molecules and the method of preparation of the same. The resultant kit is useful as a tool for phenotypic to genotypic throughput assaying of investigational drug candidates.

BACKGROUND AND PRIOR ART DISCUSSION

A central challenge for research in regenerative medicines is to identify and develop drugs that can help in curing the degenerative diseases. Lack of suitable human cell-based in vitro system that resemble progenitor stem cells which are capable of differentiating into various cell types such as neurons, myocytes, beta-cells, osteocyts, chondrocytes, adipocytes, hepatocytes and epithelial cells etc. has decelerated the very discovery process with identified effect on human simulated system of these new druggable molecules.

Identifying and developing new and useful druggable molecules continue to guzzle lots of technical and financial resources. It is always difficult for identifying new and useful drug therapies for degenerating pathologies. The improvement in treatment and increasing a patient's survival will be relied on the development of targeted and effective therapies. Further a therapy for treating age-related degenerating diseases can improve a person's quality life and slowing down the process of degeneration. Therefore identifying the right kind of druggable compounds and screening them for their biological relevance requires art that may be effective in such regiments.

As years pass by, the pharmaceutical companies stressed on incremental improvements in research of existing drugs, the repetitive rounds have resulted in a large percentage of the available drugs directed to similar targets of the modified compound and biological testing. The emphasis of pharmaceutical research activities began moving towards the purposeful discovery of novel chemical classes and novel molecular targets years ago. The birth of high throughput screening or HTS actually took place with the change in emphasis and timely technological breakthroughs, which is now widespread throughout the biopharmaceutical industry.

Creating a patient or donor derived progenitor cell based assay that is predictive of a particular physiological response; automating the assay so that it can be reproducibly performed a large number of times; and sequentially testing samples from a chemical library to identify chemical structures able to “hit” the assay, are the steps involved in screening, suggesting that such structures might be capable of provoking the intended physiological response. To eliminate artifactual results, particularly toxic compounds high throughput screen are followed in a variety of secondary assays.

To detect the presence of chemical samples possessing biological or biochemical properties high throughput screens are intended. These properties are chosen to identify compounds with the potential to elicit a specific biological response when applied in vivo. Drug candidates are identified in high throughput screens rather than the agents that will ultimately be used as drugs. A level of desired biological property in a high throughput assay can be the basis for synthesis of derivative compounds by medicinal chemists that is found from the compound of a certain chemical class.

US20160109430 entitled “Methods for drug discovery” discloses assays for identifying drug candidates that regulate cellular senescence and it is based on the discovery that the number of ATRX foci increases in cells that undergo senescence. Accordingly, the cited invention provides for assays and kits for identifying drug combinations that may be useful in treating subject that have cancers and for identifying compounds that may be useful in treating degenerative diseases like Alzheimer's, Parkinson's, Amyotrophic Lateral Sclerosis, Osteoarthristis, Degenerating Cartilage disorder, Diabetes, Muscular dystrophies and also for Breast augmentation, Myocardial muscle regeneration.

US20160209398 entitled “Assay for drug discovery based on in-vitro differentiated cells” discloses an assay system for determining therapeutic or toxic effect of a putative drug based on assaying its activity in cells which have been differentiated in vitro from stem cells, and induced to display a phenotype that resembles a disease to be treated.

However, there are no human specific cell based assay kits available to actually verify or predict the differentiation capacity of either a known drug molecule or a completely new chemical library with unknown biological relevance that can be tested properly on cultured and well defined stem cells in vitro as effectively as the present invention. The phenotypic changes of the cells under the exposure of molecules can be observed in as early as 24 hours to 28 days which is an evidence for the greater efficiency of this cell based assay kit as disclosed in the forthcoming paragraphs.

SUMMARY OF THE INVENTION

The main objective of the present invention is to exploit the abundant stromal source of mesenchymal stem cells, it's pluripotent nature to develop a homogenous cell based platform that would predict the trans-differentiation capabilities of small molecules when exposed to the cell-based platform. The prepared platform has the power to convert into neurons, myocytes, beta-cells, osteocytes, chondrocytes, adipocytes, hepatocytes and epithelial cells upon induction with the small molecule. The present invention also provides a novel method of preparation of the platform (kit) that is eligible for small molecule exposure and is compatible with the solvent in which small molecules are dissolved to transform into special cell type directing the small molecule's inducing capacity that indirectly predicts its regenerating or differentiation role in disease where the specific cell type is damaged.

BRIEF DESCRIPTION OF THE DRAWINGS

The above-mentioned and other features and advantages of this present disclosure, and the manner of attaining them, will become more apparent and the present disclosure will be better understood by reference to the following description of embodiments of the present disclosure taken in conjunction with the accompanying drawings, wherein:

Figure 1 shows a perspective view of the predictive platform as described in the present invention including plurality of wells comprising the respective differentiator, growth medium and positive controls; and

Figure 2 shows phenotypic pictographs illustrating fibroblastic mesenchymal stem cell morphology changing to neuronal processes as per the experiment conducted in example 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the exemplary embodiment(s) of the system. Before describing in detail embodiments that are in accordance with the present disclosure, it should be observed that the embodiments reside primarily in combinations of components of the platform.

In this document, the terms "comprises," "comprising," or “including” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a system, method, platform, article, device or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such system, method, article, device, or apparatus. An element proceeded by "comprises ...a" does not,
without more constraints, preclude the existence of additional identical elements in the process, product, method, article, device or apparatus that comprises the element.

Any embodiment described herein is not necessarily to be construed as preferred or advantageous over other embodiments. All of the embodiments described in this detailed description are illustrative, and provided to enable persons skilled in the art to make or use the disclosure and not to limit the scope of the disclosure, which is defined by the claims.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit and scope of the invention. Thus it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

In the following description, for the purpose of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present construction of a novel device. It will be apparent, however, to one skilled in the art that the present invention can be practiced without these specific details.

The clinical research conducted in this invention particularly concentrates on the MSCs derived from umbilical cord, dental pulp and other stromal mesenchymal tissues. This method does not involve surgery or any invasive operational activities to collect any tissues; rather, they are collected form the discarded biological waste thrown into the garbage or as a traditional discard. The MSCs from the above mentioned mesenchymal tissues are isolated, cultured and expanded by the novel method as mentioned in the parent application 932/CHE/2013.
The passage 1 homogenous characterized cells obtained from the method as mentioned in 932/CHE/2013 were seeded (5,000 cells/well) in 100 µl of complete medium (DMEM with 15% fetal bovine serum and 1% penicillin/streptomycin) in a 12-welled plate and grown overnight at 37°C; 5% CO2 and 95% humidity. Following the overnight incubation, the growth media is aspirated and the marked wells were added with freshly prepared induction media as labelled with positive controls and test compounds. The plate is re-incubated at 37°C, 5% CO2 incubator as shown in Figure 1. The media is freshly fed every 72 hr by discarding the spent one for 28 days. The MSCs harvested from the patient's bone marrow and venous blood were also passaged once and seeded as described above for the assay kit.

Assay Kit:

In non-limiting embodiments, the present invention provides for a cell-based assay kit for determining the differentiation capacity and functional property of a molecule which is mainly applied in the field of drug discovery. A cell-based assay kit predicting the cell differentiation capacity and functionality of any small molecule, comprises of a culturing plate with multiple wells coated with active progenitor mesenchymal stem cells; a growth medium; plurality of differentiation media; a positive control suitable for such differentiation being evaluated; and a means for predicting the functionality of the test molecule.

Table 1 below shows the list of growth media and differentiation media available along with the assay kit and Table 2 shows the list of positive controls available for the respective differentiation.

Table I

Particulars Available sample in the kit
Growth medium Dulbecco's Minimum Essential Medium (DMEM) and Epidermal Growth Factor
NM Neurogenic Differentiation Medium(C28015) (Promega)
AM Adipocyte Differentiation Medium (AL521) (Himedia)
KM Epithelial Cell Differentiation Medium (ax0035) (Axol)
DMSO Dimethyl Sulfoxide
OM Osteocyte Differentiation Medium (AL522) (Himedia)
PM
Beta Cell Differentiation Medium
SM1
Small molecule (10-20mM in DMSO)
M Cardio Myogenic Differentiation Medium( A25042SA ) (Gibco)

:

Table 2

Positive controls Purpose
Forskolin (Tocris) Neurogenic Differentiation
Ascorbic acid (Lonza) Adipogenic Differentiation
Valproic acid (Sigma) Epithelial Cell Differentiation
Quercetin Hydrate (TCI chemicals) Osteocyte Differentiation Medium
5-Iodotubercidin (Sigma) Beta Cell Differentiation Medium
Phorbol myristate acetate (Sigma) Cardio Myogenic Differentiation

Example 1:

The predictive platform is treated with small molecule X @ 10mM in sterile DMSO/well labelled cells + SM1 and cells + SM2 wherein SM1 is small molecule 1 and SM2 is small molecule 2. The wells are freshly supplemented with 10mM of small molecule X for every 72 hr. The phenotype pictographs as presented in Figure 2 show fibroblastic mesenchymal stem cell morphology changing to neuronal processes as early as day 3 while the positive control well with forskolin showed the differentiation into neuronal morphology after 9th day (data not shown).