Specification
FIELD OF THE INVENTION
The present invention relates to field of stem cell research. It relates to the use of Swama bhasma or gold nanoparticles in perpetuating sternness of stem cells
BACKGROUND AND PRIOR ART OF THE INVENTION
Stem cells are associated with tissue formation during embryogenesis and in their repair and maintenance during injuries and aging in the adults. These cells are unique in their capacity to 1) self-renewal, making more copies of itself and 2) retain potential to differentiate into multiple (multipotent) or all the germ lineages, pluripotency (1,2,3). These properties which distinguish them from other cell types are collectively defined as the "sternness" of these cells (4, 5). Embryonic stem cells (ESC) are derived from the inner cell mass of a peri-implantation embryo and are known to be pluripotent (Deb et al., 2008). During subsequent development towards formation of a complete organism, the cells of the embryo chronologically progress through proliferation, lineage commitment, lineage progression, lineage expression, cellular inhibition and regulated apoptosis resulting in the formation and maintenance of the differentiated cells, tissues, and organs in an individual (6,7). Although most cells advance through this sequence during development, a few cells leave the developmental continuum to become reserve precursor cells in the adult tissue (7, 8). There are two categories of these precursor cells namely lineage-committed or multipotent, somatic stem cells and lineage-uncommitted pluripotent stem cells known as the embryonic like stem cells (ELSCs). These reserve precursor cells incessantly maintain and repair tissues and organs throughout the life span of the organism (9, 10, 11). It is also believed that owing to their pluripotent capacity, ELSCs are the most potent tissue resident stem cells, and activation of this population may lead to the best possible tissue regeneration (12, 13). Moreover, it has been reported that the tissue resident ELSCs, for example of muscle tissue, can also get mobilized to the peripheral blood after trauma (13). Further, Kucia et al (14) demonstrated the mobilization of human adult-derived ELSCs into the circulatory system after stroke (15).
The use of gold and gold compounds for therapy in ancient Greek, Arabic, Chinese and Indian systems of medicine and in modern medicine has been reviewed and discussed at length in literature (16-23). In the 5000 year old Indian traditional medicine, Swama
bhasma (SB) meaning gold ash, has been extensively used for treating various clinical disorders like bronchial asthma, rheumatoid arthritis, diabetes mellitus, nervous diseases, etc. (Kean et al., 1985; Zhao and Ning 2001).
Swama Bhasma, a traditional mixture was recently physiochemically characterized and studied for anti-arthritic activities (Brown CL et al, gold bulletin 2007). Brown et al., (2007) showed that the gold particles in these SB preparations were zerovalent and of average 27 ±3 nm in size, have properties similar to that of modem day nanoparticles. In an earlier study, employing X-ray diffraction (XRD) analysis. Brown CL et al (2007) showed that the SB preparations were free of organic compounds and mainly consisted of standard gold metal (Au°). Their results also indicated that the average size of the particles were about 57 nm and had a globular morphology.
The most active area of research in gold based pharmaceuticals at present is in its application as anti-tumor agents (22, 25-29). Several other metals are also used in the Indian traditional medicine after converting them into oxides or sulfides mainly to eliminate their reactivity. Metallic gold (Au ), which is practically non-reactive or inert anyway, only needs to be sufficiently small particles, enabling them to circulate in the system and exert their effects much longer than ordinary medicines can. SB is given orally to patients, usually mixed with honey. It is believed that, in this way the gold particles which are often of nano size, and expected to have attained nanoparticulate properties, get absorbed through sublingual route directly into blood stream like a homeopathic drug (Brown CL et al., 2007 Gold bulletin). Another recent study by Hillyer and Albrecht (2006) demonstrated that gold nanoparticles smaller than 58 nm in size is absorbed in the small intestine and reach various organs through blood. In the recent years, nanoparticles of gold have attracted tremendous attention for their applications in medicine (25, 26, 30).
Human embryonic stem cells (hESCs) recapitulate organogenesis when they differentiate into tissues of various lineages. They have been widely used as suitable tools to study developmental toxicity and as a model source for understanding the molecular mechanisms of pluripotency (Sivasubramaniyan et al., 2008). The in vitro properties of the hESC make them attractive targets for research and therapy (Deb and Sarda, JTM, 2008). They provide an unlimited source of any cell type owing to their
capacity to sustain pluripotency. However, the greatest draw back of maintaining hESCs in culture is their tendency to differentiate spontaneously (31). In the most commonly practiced hESCs cultures, which involve FGF-2 supplementation to sustain pluripotency, the hESCs need to be passaged on every third-fourth day to maintain their characteristics which is expensive and time consuming (32). The hESCs spontaneously differentiate and tend to loose their proliferation capacity in culture from day 4 onwards, thereby loosing their "sternness" over time. This presents an unprecedented in vitro system to test the effect of growth factors and compounds on maintenance of pluripotency and self renewal.
Several groups have also reported about biological additives (growth factors and various pathway inhibitors) that can improve hESC culture conditions and reduced spontaneous differentiation of hESCs in cultures (references Ludwig et al., 2006 (a) and (b) nat methods and nat biotech).
The present invention overcomes the problem associated with the prior art mentioned above.
OBJECTS OF THE INVENTION
The main object of the present invention is to develop a method to perpetuate stemness of stem cells using gold particles or Swama bhasma.
Another object of the present invention is to obtain gold nanoparticle (GNP) at concentration of about 0.1)Lig/ml to about 20fxg /ml for perpetuating stemness of stem cells.
Yet another object of the present invention is to obtain a composition comprising fibroblast growth factor alongwith gold particles or Swama bhasma.
Still another object of the present invention is to obtain a medium for perpetuating stemness of stem cells, said medium comprising components of ES media alongwith gold particle or Swama Bhasma.
Still another object of the present invention is to obtain a composition comprising Swarna bhasma and gold particles.
STATEMENT OF THE INVENTION
Accordingly, the present invention relates to gold nanoparticle (GNP) at concentration of about 0.1|xg/ml to about 20|a,g /ml for perpetuating stemness of stem cells; a method to perpetuate stemness of stem cells comprising step of growing the stem cells in presence of gold particle or Swarna Bhasma; a composition for enhancing pluripotency of stem cells, said composition comprising Fibroblast Growth Factor alongwith gold particles or Swama Bhasma; a medium for perpetuating stemness of stem cells, said medium comprising components of ES media alongwith gold particle or Swama Bhasma; and a composition for perpetuating stemness of stem cells, said composition comprising Swarna Bhasma and gold particles, preferably gold nanoparticles.
BRIEF DESCRIPTION OF ACCOMPANYING FIGURES;
Figla: Graph showing size distribution by volume of the SB preparation Fig lb: Graph showing size distribution by volume of the GNP preparation Fig Ic: Transmission Electron Microscopy picture of GNP Fig Id: Transmission Electron Microscopy picture of SB
Fig 2a:Showing the effect of different doses of SB preparation on HUES9 grown in
normal hESC media, by MTT assay.
Fig 2b: Showing the effect of different doses of GNP on proliferation and pluripotency
ofhESCs.
Fig 2c: Karyotype of HUES9 PI5 grown in the presence of SB
Fig 3a: QRT-PCR for ABCG2 in day 8 SB treated samples Fig 3b: QRT-PCR for ABCG2, Oct4 and Nanog in day 5 GNP treated samples Fig 3c: QRT-PCR for ABCG2, Oct4 and Nanog in day 5 SB treated samples Fig 3d: QRT-PCR for ABCG2, Oct4 and Nanog in day 8 GNP treated samples
Fig 4a: RT-PCR analysis for the expression of: 1) pluripotency markers (0CT4 and NANOG); and 2) lineage markers a)ectoderm (P III Tubulin, Nestin); b) endoderm (GATA4, AFP); and c) mesoderm (Brachury, BMP2) in HUES? & 9 cells grown in the absence (hES-C), presence of gold bhasma ( hES-SB) and presence of gold nanoparticles ( hES-GNP) and in EBs formed from HUES9 grown in the absence and
presence of Au bhasma. Day 5 & 8 hES-C and hES-GNP showed the presence of most of the lineage markers but Day5 and Day8 hES-SB showed the absence of all other lineage markers except ectoderm genes.The EBs formed from hES-C, hES-SB and hES-GNP showed expression for all the lineage markers.
Fig 4b: Immunolocalization of Oct4 and SSEA4in HUES9. Panels (a) and (c) show the localization of Oct4 and SSEA4 in control respectively (b) and (d) show the localization of Oct4 and SSEA4 in presence of SB respectively, (c) and (f) show the localization of Oct4 and SSEA4 in presence of GNP respectively.
Fig 5: Graphs showing cell proliferation after SB (lug/ml) and GNP (lOug/ml) supplementation.
Fig 6: Phase contrast pictures of Day 4HUES-9 colonies and embryoid bodies (EBs). Panel (a) shows control HUES9 colonies. Panels (b) and (c) show morphology of undifferentiated HUES9 colonies growing on MEF in the presence of SB and GNP respectively. Panel (d) shows normal day 5 EBs. Panels (e) and (f) shows morphologies of Day5 EBs induced from HUES9 grown in the presence of SB and GNP, respectively.
Fig 7: Semiquantitative RT-PCR analysis of the expression of FGFRl and IGF2 in day 8 HUES9 cells grown in the absence and presence of gold bhasma.
Fig 8: Differential uptake of SB and GNP by embryonic and fibroblast niche cells.
Fig 9: FACS analyses of the expression of Tra 1-60 in day 8 HUES9 cells grown in the absence and presence of SB or GNP plus FGF2.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to gold nanoparticle (GNP) at concentration of about 0.1)ig/ml to about 20^g /ml for perpetuating stemness of stem cells.
In another embodiment of the present invention, the concentration of GNP is preferably about 10|ig/ml.
In yet another embodiment of the present invention, the GNP has diameter of about I5nm to about 16.5nm.
In still another embodiment of the present invention, the stem cells are lineage-uncommitted pluripotent stem cells.
The present invention relates to a method to perpetuate stemness of stem cells comprising step of growing the stem cells in presence of gold particle or Swarna Bhasma.
In still another embodiment of the present invention, the method perpetuates stemness by enhancing proliferation, self-renewal and pluripotency and reducing spontaneous differentiation of the stem cells.
In still another embodiment of the present invention, the gold particle is gold nanoparticle (GNP).
In still another embodiment of the present invention, the gold nanoparticle (GNP) is present at a concentration of about 0.1|j,g/ml to about 20|xg/ml, preferably about lOng/ml. In still another embodiment of the present invention, the Swarna bhasma is present at a concentration of about 0.1|ig/ml to about 2|Ag / ml, preferably about l(ig/ml.
In still another embodiment of the present invention, the stem cells are lineage-uncommitted pluripotent stem cells.
The present invention also relates to a composition for enhancing pluripotency of stem cells, said composition comprising Fibroblast Growth Factor alongwith gold particles or Swarna Bhasma.
In still another embodiment of the present invention, the gold particle is gold nanoparticle (GNP).
In still another embodiment of the present invention, the GNP is present at a concentration of about 0.1|a.g/ml to about 20\ig I ml, preferably about lOng/ml.
In still another embodiment of the present invention, the GNP has diameter of about 15nm to about I6.5nm.
In still another embodiment of the present invention, the Swama bhasma is present at a concentration of about 0.1|ig/ml to about 2\ig I ml, preferably about I|j,g/ml.
In still another embodiment of the present invention, the Fibroblast Growth Factor is a basic fibroblast growth factor or FGF2.
In still another embodiment of the present invention, the FGF2 is present at a concentration of about 2ng/ml to about 40ng/ml, preferably about 4ng/ml.
In still another embodiment of the present invention, the stem cells are lineage-uncommitted pluripotent stem cells.
The present invention also relates to a medium for perpetuating sternness of stem cells, said medium comprising components of ES media alongwith gold particle or Swarna Bhasma.
In still another embodiment of the present invention, the gold particle is gold nanoparticle (GNP).
In still another embodiment of the present invention, the gold nanoparticle (GNP) is present at concentration of about 0.1|j,g/ml to about 20ng / ml, preferably about lO^g/ml.
In still another embodiment of the present invention, the GNP has diameter of about 15nm to about 16.5nm, preferably about 15.59nm.
In still another embodiment of the present invention, the Swama bhasma is present at a concentration of about 0.1 |Ag/ml to about 2\ig I ml, preferably about 1 (ig/ml.
In still another embodiment of the present invention, the stem cells are lineage-uncommitted pluripotent stem cells.
In still another embodiment of the present invention, the components of ES media include 80% Knockout DMEM, 20% Knockout serum replacer, L-glutamine at concentration of about 2mM, 1% nonessential aminoacids, P-mercaptoethanol at concentration of about O.ImM, human FGF2 at concentration of about 4ng/ml and penicillin streptomycin at concentration of about 50U/ml.
The present invention also relates to a composition for perpetuating sternness of stem cells, said composition comprising Swarna Bhasma or gold particles, preferably gold nanoparticles.
The present invention relates to gold particulate preparation on improvement of the stem cell proliferation, self renewal and pluripotency. The gold particulate preparation, traditionally known as Swarna bhasma (SB) was adapted and prepared by the methods described in traditional Indian medicinal literature.
Trace element analyses of the SB preparations by inductively coupled plasma-optical emission spectrometry (ICPOES) showed no statistically significant variation in the trace elements compositions, measured in parts per million (ppm), across samples obtained from different batches. The presence of mercury and lead less than the minimal detection levels in the aforesaid preparations indicated a high quality product with lesser toxicity and better potential acceptability as a medicine.
With Dynamic light scattering studies, it was observed that more than 70% of the particles were of approximately 41 nm in size. Since, gold in the SB was found to be
smaller than 58 nm in size, it is envisioned that they reach the tissues through blood even after oral administration.
In the present invention, the hESC cultures were supplemented with SB in presence and absence of FGF-2 to determine its effect on pluripotency. We found that SB supplemented at a concentration of lug/ml supported increased cell proliferation and also increased the expression of pluripotency markers like ABCG2, as compared to the control. This dose also reduced spontaneous differentiation of the ESCs till day 8, without compromising their ability to give rise to cells of the three germ layer lineages. However, the SB particles exhibited their best effect up to day 8 as the cells showed complete silencing for most lineage markers.
Since SB is predominantly comprised of metallic inert gold (Au°) nanoparticles, the role of synthesized colloidal gold (Au") nanoparticles (GNP) on the stemness of the cells was assessed. It was found that the expression of pluripotency genes like ABCG2 were upregulated significantly over the control. Fig 8 provides the uptake of GNP's and SB by hES cells.
Intriguingly, this study also indicates that gold supplementation to the hESC culture media along with FGF-2 helps in improving the fidelity of the cultures and in sustaining prolonged pluripotency. Human ESC cultures are known to be susceptible to karyotypic abnormalities like trisomy 12 and / or 17 due to selection pressure during cultures over several passages. Therefore, it was imperative to confirm that the GNPs and the SB preparations did not negotiate the pluripotency and chromosomal stability of the ESCs even upto 15 passages (Fig 2c).
Further, the present invention proves that gold brings about this effect by improving the niche population. The pool of niche cells in hESC is known to secrete IGF-2 and express FGFRl. The secreted IGF-2 in turn was shown to facilitate maintainance of pluripotency in hESCs. Likewise, an upregulation in the expression of FGFRl and IGF-2 was detected. Further, an increase in the proliferation of the total population of cells in the SB treated culture plates from day 4 onwards was observed. These results implicate that SB preparations improve stemness by inducing the expression of IGF-2
in the niche cells without altering the ratio of niche cells in an ESC microenvironment. It appears obvious that due to an overall increase in the niche cell pool in the cultures, the total amount of secreted IGF-2 in the culture media is proportionately up-modulated.
Both SB and GNP when used in combination with FGF-2, significantly upregulated the expressions of pluripotency markers by day 5 and hence it is apparent that the gold nanoparticles in SB or GNP are the main players orchestrating this effect.
Another important aspect of any culture media supplements is their interference in the freeze-thaw efficiencies of the cells. Several media components are known to selectively improve or affect the freeze-thawing efficiencies of cells. Our preliminary studies indicate no adverse effect on the efficacy of the cells to revive after being cultured continuously in presence of SB or GNP over several passages. These findings strongly indicate that gold nanoparticles or SB at its determined dose can be supplemented in hESC cultures to improve hESC pluripotency. Their potential to alleviate the proliferation, pluripotency and self renewal in ESCs suggest that they could possibly bring about the same effects in vivo in the tissue resident pluripotent population of ELSCs. The study therefore provides an in vitro evidence for the function of gold preparations in enhancing rejuvenation and regeneration through hESC.
The invention is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of invention.
Example 1: Human embryonic stem cell (hESC) cultures and in vitro maintenance:
hESC line HUES9 and HUES? were obtained from Douglas Melton at Harvard University. They were grown on mitomycin-C-treated CFl mouse derived embryonic feeder (MEFs) ceils at 37°C, 5% C02 in the ES media which consists of 80% KO DMEM, 20% Knockout serum replacement, 2mIVl L-Glutamine, 1% nonessential amino acid solution, 0.1 mM 6-mercaptoethanol, 4ng/ml human FGF2 and 50U/ml pencil I in-streptomycin (all from Invitrogen, CA, USA). The cells were passaged every 4 days to maintain pluripotency and selfrenewal.
Feeder-free undifferentiated HuES9 human ES cells were maintained on Matrigel (1:15 dilution, Sigma) coated dishes in conditioned medium containing knockout DMEM/20% serum replacement, 2mM L-Glutamine, 1% nonessential amino acid solution, 0.1 mM 6-mercaptoethanol and 50U/ml pencillin-streptomycin (ail from Invitrogen, CA, USA). Conditioned medium was obtained by culturing mouse embryonic fibroblast (MEF) cells with HuES9 media. The medium was collected at 24 h intervals, filter sterilized and further supplemented with 8 ng/ml bFGF for HuES9 cell culture.
Example 2: Swarna Bhasma (SB) preparation:
One part of gold and eight parts of mercury were taken together in a mortar and amalgam was prepared by continuous trituration for 3 days. To this, sixteen parts of sulfur was added and triturated till homogenous mixture is obtained. This mixture was then taken in a mud smeared beer bottle and kept in the furnace in a specially designed vessel called "valuka yantra" (a metal bucket filled with coarse and uniform gravels of sand). Continuous and controlled heat was given for about 6-8 hrs. The bottle in the furnace was left for self cooling. The bottle was cut open in the middle and product from the bottom of the bottle was collected. The product from the bottom of the bottle was mixed well with equal parts of white arsenic (AS2O3) and impregnated with juice of Ocimum sanctum. Now the contents were taken in an earthen crucible .The crucible was covered by placing another inverted earthen crucible on top of it and sealed by three layers of cotton cloth and wet clay. The assembly was heated for 8h using cow dung cakes (eight cakes) in a pit. The maximum temperature during heating was ~900°C. This was opened on the next day and product was collected. This was again mixed with half part of white arsenic and again impregnated with juice of Ocimum sanctum. Then it was kept in a crucible and ignited. This procedure was repeated for 8-10 times or till it attained proper qualities of final product. This product was repeatedly made over a number of times and analyzed for particle size and consistency in trace element compositions and batch variations.
Gold nanoparticle (GNP) preparation:
To prepare the GNPs, 20ml of ImM auric chloride was heated to boiling in an Erlenmeyer flask with stirring. 2ml of a 1% solution of trisodium citrate was added slowly to this boiling solution and heating was continued for about 20 min. The citrate reduces auric chloride to gold nanosuspension. The completion of the reaction was detected by the formation of deep red suspension. This was analyzed for particle size subsequently.
Example 3: Trace element analysis of SB preparations:
Concentrations of minor and trace elements (As, Cd, Hg and Pb) in the swarna bhasma (SB) were analyzed by the inductively coupled plasma-optical emission spectrometry (ICPOES) method using Perkin Elmer Optima 5300DV. lOmg of the bhasma was dissolved in 0.5 ml of Nitric acid. This was made up to 10 ml using deionised water to make a concentration of lOmg/ml and was used for ICP-OES studies. An equivalent concentration of nitric acid was used as blank. Readings were made in axial mode and trace element concentration was evaluated from the standard calibration curve. To check if the compositions of the trace elements vary with the batches of SB preparations, trace element analyses for three different sample preparations SB I, SB2 and SB3 was done.
Trace element analysis using Optical Emission Spectroscopy with Inductively Coupled Plasma of the SB preparations showed detectable amounts of arsenic (29.27 ± 0.67ppm) and cadmium (0.636 ± 0.02ppm), while the level of mercury and lead were found to be below minimum detection levels (Table 1).
Sample code Element
Arsenic (ppm) Cadmium (ppm) Mercury (ppm) Lead (ppm)
SBl 29.70 0.622
Documents
Application Documents
| # |
Name |
Date |
| 1 |
2601-CHE-2008 POWER OF ATTORNEY -14-08-2009.pdf |
2009-08-14 |
| 1 |
2601-CHE-2008_EXAMREPORT.pdf |
2016-07-02 |
| 2 |
2601-CHE-2008 AMENDED CLAIMS 05-12-2011.pdf |
2011-12-05 |
| 2 |
2601-che-2008 form-5.pdf |
2011-09-04 |
| 3 |
2601-che-2008 form-3.pdf |
2011-09-04 |
| 3 |
2601-CHE-2008 FORM-1 05-12-2011.pdf |
2011-12-05 |
| 4 |
2601-che-2008 form-18.pdf |
2011-09-04 |
| 4 |
2601-CHE-2008 FORM-13 05-12-2011.pdf |
2011-12-05 |
| 5 |
2601-che-2008 form-1.pdf |
2011-09-04 |
| 5 |
2601-CHE-2008 FORM-3 05-12-2011.pdf |
2011-12-05 |
| 6 |
2601-che-2008 drawings.pdf |
2011-09-04 |
| 6 |
2601-CHE-2008 EXAMINATION REPORT REPLY RECEIVED 05-12-2011.pdf |
2011-12-05 |
| 7 |
2601-che-2008 description (complete).pdf |
2011-09-04 |
| 7 |
2601-CHE-2008 CORRESPONDENCE OTHERS 28-09-2011.pdf |
2011-09-28 |
| 8 |
2601-che-2008 correspondence others.pdf |
2011-09-04 |
| 8 |
2601-CHE-2008 FORM-3 28-09-2011.pdf |
2011-09-28 |
| 9 |
2601-CHE-2008 POWER OF ATTORNEY 28-09-2011.pdf |
2011-09-28 |
| 9 |
2601-che-2008 claims.pdf |
2011-09-04 |
| 10 |
2601-che-2008 abstract.pdf |
2011-09-04 |
| 11 |
2601-CHE-2008 POWER OF ATTORNEY 28-09-2011.pdf |
2011-09-28 |
| 11 |
2601-che-2008 claims.pdf |
2011-09-04 |
| 12 |
2601-CHE-2008 FORM-3 28-09-2011.pdf |
2011-09-28 |
| 12 |
2601-che-2008 correspondence others.pdf |
2011-09-04 |
| 13 |
2601-CHE-2008 CORRESPONDENCE OTHERS 28-09-2011.pdf |
2011-09-28 |
| 13 |
2601-che-2008 description (complete).pdf |
2011-09-04 |
| 14 |
2601-CHE-2008 EXAMINATION REPORT REPLY RECEIVED 05-12-2011.pdf |
2011-12-05 |
| 14 |
2601-che-2008 drawings.pdf |
2011-09-04 |
| 15 |
2601-CHE-2008 FORM-3 05-12-2011.pdf |
2011-12-05 |
| 15 |
2601-che-2008 form-1.pdf |
2011-09-04 |
| 16 |
2601-CHE-2008 FORM-13 05-12-2011.pdf |
2011-12-05 |
| 16 |
2601-che-2008 form-18.pdf |
2011-09-04 |
| 17 |
2601-CHE-2008 FORM-1 05-12-2011.pdf |
2011-12-05 |
| 17 |
2601-che-2008 form-3.pdf |
2011-09-04 |
| 18 |
2601-CHE-2008 AMENDED CLAIMS 05-12-2011.pdf |
2011-12-05 |
| 18 |
2601-che-2008 form-5.pdf |
2011-09-04 |
| 19 |
2601-CHE-2008_EXAMREPORT.pdf |
2016-07-02 |
| 19 |
2601-CHE-2008 POWER OF ATTORNEY -14-08-2009.pdf |
2009-08-14 |