A method, a kit and the use of pluripotent stem cells induced using cell-free chromatin particle (cfchps)

Abstract

The invention presented herein introduces a pioneering method for reprogramming somatic cells into pluripotent states through the application of cfChPs isolated from human serum. The method comprises isolating cell-free chromatin particles (cfChPs) from human serum samples; and treating the somatic cells to induce pluripotency. Induced pluripotent stem cells (iPSCs), are characterized by up regulation of stem cell related transcription factors, namely, OCT4, SOX2, KLF4 and NANOG genes; increased expression of stem cell surface markers, namely, CD34, CD44, and CD133, formation of spheroids; and teratoma formation in mice. The innovative utilization of cfChPs for reprogramming somatic cells into iPSCs represents a transformative leap in stem cell production. This approach has the potential to revolutionize regenerative medicine, disease modeling, and drug discovery, paving the way for a new era of personalized and effective therapies.

Description

[0001] TITLE: A method, a kit and the use of pluripotent stem cells induced using cell -free chromatin particles (cfChPs)

[0002] FIELD OF THE INVENTION

[0003] The invention presented herein introduces a pioneering method for reprogramming somatic cells into pluripotent states through the application of cfChPs isolated from human serum.

[0004] DEFINITIONS

[0005] As used in the present disclosure, the following terms are generally intended to have the meaning as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0006] CfChPs refers to cell-free chromatin particles isolated from human serum.

[0007] Cell reprogramming refers to conversion or rather silencing of the somatic genes and, the reactivation of pluripotent stem cell-expressed genes to change the identity and functioning of the somatic cell.

[0008] BACKGROUND

[0009] Stem cell research has undergone decades of sustained and rigorous exploration, driven by its profound potential for a myriad of therapeutic applications within regenerative medicine. Stem cells are recognized as an invaluable asset due to their unique capacity to replace or regenerate impaired or dysfunctional cells and tissues in the human body. This inherent regenerative prowess has not only captivated the scientific community but also attracted considerable commercial investment. A reference may be made to “Zakrzewski, W., Dobrzynski, M., Szymonowicz, M. et al. Stem cells: past, present, and future. Stem Cell Res Ther 10, 68 (2019 in this respect.

[0010] A significant advancement in this field is the emergence of induced pluripotent stem cells (iPSCs), a category of pluripotent stem cells derived from adult somatic cells. The groundbreaking discovery of iPSC cells by Takahashi and Yamanaka in 2006 allowed for the generation of pluripotent stem cells via the ectopic expression of four critical genes — Oct4, Sox2, Klf4, and c-Myc — in both embryonic and adult murine fibroblasts, without the ethical concerns associated with embryonic use, as refenced in “Takahashi K, Yamanaka S et.al Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors." Cell. 2006; 126(4): 663 -16” The approach offered a powerful means to "reprogram" cells whose developmental destinies were traditionally thought to be predetermined.

[0011] Notably, iPSC cells are characterized by the expression of surface stem cell markers (such as CD34, CD 133, Tra-1-60, and SSEA-1), the ability to form spheroids in-vitro and teratomas in-vivo, and the potential to differentiate into diverse tissue types when injected in severely immune deficient (SCID) mice as reported n “Baghbaderani BA, Syama A, Sivapatham R, Pei Y, Mukherjee O, Fellner T, Zeng X, Rao MS. Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications. Stem Cell Rev Rep. 2016 Aug; 12(4):394-420” and “Wesselschmidt RL. The teratoma assay: an in vivo assessment of pluripotency, Methods Mol Biol. 2011; 767:231-41”. Moreover, tissues derived from iPSC cells closely resemble the injected donor cells, making them invaluable for disease modeling and drug screening is reported in “Nicholson MW, Ting CY, Chan DZH, Cheng YC, Lee YC, Hsu CC, Huang CY, Hsieh PCH. Utility of iPSC-Derived Cells for Disease Modeling, Drug Development, and Cell Therapy. Cells. 2022 Jun 6;11(11):1853” It is anticipated that iPSC cells will enable the reprogramming of cells to restore damaged tissues in the human body, as mentioned in “Ye L, Swingen C, Zhang J. Induced pluripotent stem cells and their potential for basic and clinical sciences. Curr Cardiol Rev. 2013 Feb l;9(l):63-72” .

[0012] Nonetheless, challenges persist in the production of iPSC cells via the ectopic expression of four critical genes also known as Yamanaka Factors. Retroviruses employed for generating iPSC cells are associated with cancer risk due to their ability to insert DNA into the genome and potentially activate cancer-causing genes disclosed in “Shao L, Wu WS. Gene-delivery systems for iPS cell generation. Expert Opin Biol Ther. 2010 TMC01

[0013] Feb;10(2):231-42” Additionally, the reprogramming efficiency of somatic cells into iPSC cells via transcription factors remains notably low, at less than 0.02%. Furthermore, the method is cost-prohibitive and slow. A reference may be made to “Doss MX, Sachinidis A. Current Challenges of iPSC-Based Disease Modeling and Therapeutic Implications. Cells. 2019 Apr 30;8(5):403”.

[0014] In efforts to overcome these challenges, several strategies have been reported. Overexpression of the epigenetic reader BRD3R, alongside three of the Yamanaka factors (OCT4, SOX2, KLF4), has been reported to increase reprogramming efficiency by promoting mitosis and resetting cell cycle profiles in human cells as mentioned in “Shao, Z., Zhang, R., Khodadadi- Jamayran, A. et al. The acetyl lysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis. Nat Commun 7, 10869 (2016)”. Another report showed that the addition of NANOG and LIN28 to the classic OSKM (OCT4, SOX2, KLF4 and cMyc) factors enhances reprogramming efficiency (~76-fold) and, shortens latency, acting through modulation of WNT and LIN41 pathways is disclosed in “Wang L, Su Y, Huang C, Yin Y, Chu A, Knupp A, Tang Y. NANOG and LIN28 dramatically improve human cell reprogramming by modulating LIN41 and canonical WNT activities. Biol Open. 2019 Dec 5;8(12):bio047225” .

[0015] Fibromodulin (FMOD), an extracellular matrix protein, was reported to reprogram fibroblasts into multipotent Fibromodulin-reprogrammed (FreP) cells. These cells were shown to differentiate into all three germ layers in vitro and regenerate muscle and bone in vivo. However, they failed to form teratomas and exhibited slow proliferation rate in the undifferentiated state, indicating incomplete pluripotency. Same is mentioned in “Zheng Z, Jian J, Zhang X, Zara JN, Yin W, Chiang M, Liu Y, Wang J, Pang S, Ting K, Soo C. Reprogramming of human fibroblasts into multipotent cells with a single ECM proteoglycan, fibromodulin. Biomaterials. 2012 Aug;33(24):5821-31” . Therefore, although, these non-genetic methods reduced the risk of genomic alterations, their reprogramming efficiency and scalability remain limited.

[0016] Human keratinocytes may also be reprogrammed into iPSCs using established techniques involving transcription factors delivered by non-integrative viral vectors or TMC01 episomal plasmids. Keratinocytes, especially those isolated from hair follicles or skin biopsies, are considered ideal starting cells for iPSC generation due to their proliferative capacity and high reprogramming efficiency compared to other somatic cell type. However, Human keratinocytes have limitations for clinical and research use, most of them related to efficiency, safety, and genetic stability. The iPSCs, including those derived from keratinocytes, are prone to tumorigenicity. The transcription factor genes used for reprogramming (e.g., c-MYC, KLF4, SOX2, OCT4) are associated with oncogenic risks. A reference may be made to “Medvedev SP, Shevchenko Al, Zakian SM. Induced Pluripotent Stem Cells: Problems and Advantages when Applying them in Regenerative Medicine. Acta Naturae. 2010 Jul;2(2): 18-28” . The Keratinocytes and their iPSC derivatives may experience limited proliferative capacity and senescence during long-term expansion, impacting their use for gene therapy and cell-based therapies. The efficiency of reprogramming keratinocytes to iPSCs varies and may require precise timing and balance of factor expression, with even small procedural differences affecting outcomes.

[0017] To address the limitations of current methods, a promising avenue lies in usingcfChPs released from dying cells into the blood circulation. In this context, a refence may be made to “I. Mittra et al., “Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes,” J. Biosci., vol. 40, no. 1, pp. 91-111, Mar. 2015”. In the human body, billions of cells undergo apoptotic cell death daily as mentioned in “R. Sender, R. Milo., “The distribution of cellular turnover in the human body, ” Nat Med. Vol. 27, pp. 45- -48, 2021”. These apoptotic cells release their chromosomal fragments in the form of cfChPs into the blood circulation leading to elevated levels of cfChPs in the bloodstream as mentioned in “K. Aoki, S. Satoi, S. Harada, S. Uchida, Y. Iwasa, and J. Ikenouchi, “Coordinated changes in cell membrane and cytoplasm during maturation of apoptotic bleb, ” Mol. Biol. Cell, vol. 31, no. 8, pp. 833 844, Apr. 2020” . Our previous studies suggest that cfChPs represent endogenous DNA-damaging agents with ongoing physiological activity, offering insights into aging and various human diseases, including cancer development, by oncogenic transformation of NIH3T3 cells and disclosed in “Raghuram GV, Chaudhary S, Johari S, Mittra I. Illegitimate and Repeated Genomic Integration of Cell-Free Chromatin in the Aetiology of Somatic Mosaicism, Ageing, TMC01

[0018] Chronic Diseases and Cancer. Genes (Basel). 2019 May 28; 10(6):407 Since cancer cells are closely linked to stem cell properties, these observations provide a compelling rationale for investigating the role of cfChPs in reprogramming somatic cells into stem cells.

[0019] There is, therefore, felt a need that mitigates the drawbacks mentioned hereinabove or at least provide a suitable alternative.

[0020] OBJECT OF THE INVENTION

[0021] The main object of the invention is to provide method toobtainiPSCsfrom somatic cells using cfChPs isolated from human serum.

[0022] Another object of the invention is to provide a method for obtaining Induced pluripotent stem cells (iPSCs) treating the somatic cells selected from NIH3T3 mouse fibroblast cells and human keratinocytes, with the cfChPs to induce pluripotency, followed by characterisation of the iPSCs.

[0023] Another object of the invention is to provide a method for obtainingiPSCs— characterised by an increase in the expression of OCT4, SOX2, KLF4 and NANOG genes; increased expression of stem cell surface markers selected from CD34, CD44, and CD133; formation of spheroids; and teratoma formation.

[0024] Yet another objective of the invention is to iPSCs production both economically viable and rapid.

[0025] Another object of the invention is to provide a method for obtaining Induced pluripotent stem cells (iPSCs), characterised by an increase in the activationof OCT4, SOX2, KLF4 and NANOG genes; increased expression of stem cell surface markers selected from CD34, CD44, and CD133; formation of spheroids; and teratoma formation.

[0026] Yet another objective of the invention is to develop iPSCs without relying on retroviruses, thereby eliminating associated cancer risks. TMC01

[0027] Yet another objective of the invention is to boost the reprogramming efficiency of somatic cells into iPSCs.

[0028] SUMMARY

[0029] The present disclosure relates to a method, a kit and the use of pluripotent stem cells obtained using cfChPs. The method for obtaining iPSCscomprising the step of isolating cfChPs from human serum samples. Further, treating somatic cells, selected from NIH3T3 mouse fibroblast cells and human keratinocytes, with cfChPs to induce pluripotency. The induction medium is Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% bovine calf serum (BCS), wherein the somatic cells are the NIH3T3 cells; and the induction medium is foetal bovine serum (FBS) wherein the somatic cells are human keratinocytes; additionally, 1% antibiotic and anti-mycotic solution. The cells were cultured in a controlled environment in an incubator at 37 °C with 5% CO2. NIH3T3 cells were treated with 10 ng of cfChPs isolated usinga previously published standardmethod described under example 7) [I. Mittra et al., “Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes,” J. Biosci., vol. 40, no. 1, pp. 91-111, Mar. 2015, while human keratinocytes were treated with 250 pg of cfChPs isolated by ChromaFlash™ Chromatin Extraction Kit (Epigentek) with some modifications (described under example 3). The iPSCs characterization involved evaluating activation of pluripotency -related genes (OCT4, SOX2, KLF4 and NANOG) and surface markers (CD34, CD44 and CD 133), spheroid formation and teratoma formation in mice.

[0030] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING

[0031] The method of the present disclosure will now be described with the help of the accompanying drawing, in which:

[0032] Figure 1 illustrates stem cell-related transcription factors gene expression in NIH3T3 cells treated with cfChPs. TMC01

[0033] Legend: Expression of stem cell transcription factors in NIH3T3 cells across passages following cfChPs treatment. (A) NIH3T3 cells treated with healthy donor- derived cfChPs (H-cfChPs) showed minimal expression of OCT4 and NANOG, with KLF4 peaking at P25. SOX2 was undetermined. (B) Cells treated with cancer patient- derived - cfChPs (C-cfChPs) exhibited higher and sustained expression of SOX2, OCT4, and NANOG with a marked KLF4 peak at PIO.

[0034] Figure 2 illustrates surface stem cell markers in NIH3T3 cells treated with cfChPs.

[0035] Legend: Passage-wise analysis of surface stem cell markers in NIH3T3 cells treated with cfChPs. (A) NIH3T3 cells treated with cfChPs derived from healthy donor serum (H-cfChPs) showed increased expression of CD44, CD34, and CD133, with a notable peak in CD44 at PIO and CD34 at P25, as measured by flow cytometry. Treatment cells showed enhanced CD44 and CD34 surface stem cell markers. (B)Cells treated with cancer patient-derived - cfChPs (C-cfChPs) also exhibited upregulation of these markers, with CD44 and CD34 showing higher and more sustained expression compared to controls, while CD133 fluctuated across passages.

[0036] Figure 3 illustrates the spheroid formation in NIH3T3 cells treated with cfChPs

[0037] Legend: cfChPs treatment induces spheroid formation in NIH3T3 cells. (A) Image of spheroids formed by NIH3T3 control cells, NIH3T3 cells 72h post- treatment of H- cfChPs, and NIH3T3 cells 72h post-treatment of C-cfChPs in Ultra-Low Attachment (ULA)-plates on 7th day after culturing in mammocult media. (B) Histograms depict the number of spheroids formed per well in 96-well ULA plates. (C) Histograms depict the results of the mean diameter of all the spheroid forms per well in 96-well ULA plates. All experiments were performed in duplicates, and box-plots represent mean ± SEM values. Statistical analyses were performed using one-way ANOVA (Graph Pad Prism 8). **** p<0.0001. TMC01

[0038] Figure 4 illustrates thatthe teratoma formation in mice.

[0039] Legend: In vivo teratoma formation and confirmation of tri -lineage differentiation in cfChPs-treated cells (A) Representative images showing teratomas formed subcutaneously in immunocompromised mice following injection of cfChPs-treated human keratinocytes. Tumor masses developed in both H-cfChPs and C-cfChPs groups, confirming in vivo tumorigenic potential. (B). Histological analysis of excised teratomas stained with H&E showing tissue organization. (C) Expression of lineage-specific markers was evaluated using IHC to confirm tri-lineage differentiation. GFAP (ectodermal marker), CD45 (mesodermal marker), and AFP (endodermal marker) were detected in teratoma sections derived from H-cfChPs and C-cfChPs treatment. Negative and positive controls are shown for comparison. Scale bars represent 50 pm.

[0040] Figure 5 illustrates stem cell-related transcription factors’ gene expression in human keratinocytes treated with H-cfChPs.

[0041] Legend- Time-course analysis of stem cell-associated transcription factors OCT4, NANOG, and KLF4 expression in human keratinocytes treated with H-cfChPs: Keratinocytes treated with cfChPs showed upregulation of mRNA expression of OCT-4, NANOG and KLF-4 peaking at 72 h as compared to untreated control cells.

[0042] Figure 6 illustrates immunofluorescence staining and quantification of transcription factorsin human keratinocytes treated with H-cfChPs.

[0043] Legend- (A) Representative images of human keratinocytestreated with H-cfChPsstained for SOX2 (red, TRITC channel) with DAPI counterstain (blue) showing nuclear morphology. Bar graphs represent mean fluorescence intensity (MFI) per cell (left) and percentage of SOX2 positive cells (right). (B)Representative images of human keratinocytestreated with H-cfChPs stained for OCT4 (red, TRITC channel), with DAPI counterstain (blue) showing nuclear morphology. Bar graphs represent mean fluorescence intensity (MFI) per cell (left) and percentage of OCT4 positive cells (right). (C)Representative images of human keratinocytes stained for NANOG (red, TRITC TMC01 channel); with DAPI counterstain (blue) showing nuclear morphology. Bar graphs represent mean fluorescence intensity (MFI) per cell (left) and percentage of NANOG- positive cells (right). Scale bar = 20 pm

[0044] Figure 7 illustrates surface stem cell markers- CD34, CD44, and CD133 in human keratinocytestreated with cfChPs.

[0045] Legend- Time-course analysis of relative differential expression (AMFI) of surface stem cell markers- CD34, CD44, and CD133 in cfChPs-treated human keratinocytes: cfChPs treatment upregulatedexpression of CD44, CD34, and CD 133 AMFI (MFI of treated -MFI of respective time -point control) as measured by flow cytometry.

[0046] Figure 8 illustrates Spheroid morphology in control and cfChPs-treated keratinocytes.

[0047] Legend- Spheroid morphology and roundness score in control and cfChPs-treated keratinocytes. Representative images of spheroids formed on day 7 under low- attachment culture conditions are shown for untreated control and cfChPs-treated (250pg) human keratinocytes. Control spheroids displayed irregular shapes, whereas cfChPs- treated spheroids were more compact and spherical. Box-and-whisker plot showing quantitative analysis of spheroid roundness scores (n = 30 spheroids per group). Data are expressed as mean ± SEM. Statistical analyses were performed using a two-tailed Student’s unpaired t-test (GraphPad Prism 8) **** p<0.0001.

[0048] Figure 9 illustrates in vivo teratoma formation and confirmation of tri-lineage differentiation in cfChPs-treated cells.

[0049] Legend- In vivo teratoma formation and confirmation of tri-lineage differentiation in cfChPs-treated cells (A) Representative image showing teratoma formed in SCID mouse post-injection of cfChPs-treated keratinocytes and gross morphology of excised teratomas from cfChPs-treated and control groups. (B) Hematoxylin and Eosin (H&E) TMC01 staining of teratoma sections from control and cfChP-treated mice (C)Immunohistochemistry (IHC) analysis for germ layer-specific markers: GFAP (ectoderm), and CD45 (mesoderm). Negative and positive controls are shown for comparison. Images captured at 20X magnification. Scale bars represent 50 pm.

[0050] DETAILED DESCRIPTION

[0051] Embodiments, of the present disclosure, will now be described with reference to the accompanying drawing.

[0052] Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

[0053] Terms such as “inner,” “outer,” "beneath," "below," "lower," "above," "upper," and the like, may be used in the present disclosure to describe relationships between different elements as depicted from the figures.

[0054] The present invention has exploited a novel approach to generate iPSCs with enhanced reprogramming efficiency, while minimising the risks of genetic modification and avoiding the use of viral vectors. The current invention is based on the use of cfChPs released from dying cells into the blood circulation. In the human body, billions of cells undergo apoptotic cell death daily these apoptotic cells release their chromosomal fragments in the form of cfChPs into the blood circulation leading to elevated levels of cfChPs in the bloodstream. TMC01

[0055] The studies suggest that cfChPs represent endogenous DNA-damaging agents with ongoing physiological activity, offering insights into aging and various human diseases, including cancer development, by oncogenic transformation of NIH3T3 cells. Since cancer cells are closely linked to stem cell properties, these observations provide a compelling rationale for investigating the role of cfChPs in reprogramming somatic cells into stem cells.keratinocyte-derived iPSCs have significant clinical and research potential, they face hurdles including tumorigenicity, genomic integration risks, limited proliferative capacity, and variability in differentiation and genetic correction. These issues must be addressed for safe and effective application in regenerative medicine and disease modeling.

[0056] The present disclosure demonstrated that cfChPs is effectively to induce iPSCs from NIH3T3 mouse fibroblast cells and human keratinocytes.

[0057] The treatment with cfChPs, these NIH3T3 cells exhibits the expression of surface stem cell markers, such as CD34 and CD 133. Moreover, these treated cells demonstrate the capacity to form spheroids when cultured on ultra-low attachment plates. Furthermore, when inoculated subcutaneously into SCID mice, these treated cells give rise to teratomas, a classic model for evaluating pluripotency. Notably, the teratomas exhibit the expression of markers representative of neural ectoderm and mesoderm germ layers, providing compelling evidence of in vivo differentiation. of the present disclosure is to provide a method for obtaining Induced pluripotent stem cells.

[0058] In an embodiment, the method comprises isolating cfChPs from human serum samples; and treating the somatic cells, with the cfChPs to induce pluripotency, followed by Characterisation of the iPSCs.

[0059] In an embodiment, the somatic cells are selected from NIH3T3 mouse fibroblast cells and human keratinocytes.

[0060] In an embodiment, the cfChPs induction activates activation of pluripotency genes, selected from OCT4, SOX2, KLF4 and NANOG. TMC01

[0061] In an embodiment, the medium is Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% bovine calf serum (BCS), wherein the somatic cells are the NIH3T3 cellsand Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10%foetal bovine serum (FBS) wherein the somatic cells are human keratinocytes, additionally, 1% antibiotic and anti-mycotic solution.The cells were cultured in a controlled environment in an incubator at 37°C with 5% CO2.

[0062] In an embodiment, NIH3T3 cells were treated with 10 ng of cfChPs isolated by a previously published standardmethod (described under example 1), while human keratinocytes were treated with 250 pg of cfChPs isolated by ChromaFlash™ Chromatin Extraction Kit (Epigentek) with some modifications (described under example 3).

[0063] In an embodiment, the standard method of Cell-free chromatin isolation comprises the steps of: i. Ultra-centrifugation of 1ml of serum at 700,000 g for 16 hours at 4°C to remove cellular debris, ii. Treatment of the pellet obtained with lysis buffer followed by ultracentrifugation at 700,000 g for 16 hours at 4°C and separation of the supernatant. iii. Suspending the pellet obtained containing cfChPs after removal of supernatant. iv. Capturing the cfChPs by passing the suspension through an affinity column containing a mixture of biotinylated anti-histone antibodies (20 pg each of Hl, H2A, H2B, H3, H4 in a volume of 1 mL) bound to Pierce® Streptavidin Plus Ultralink® Resin (Thermo Scientific, USA) (1 mL) v. Eluting bound cfChPs from the column using 0.25 M NaCl buffer vi. Ultra-centrifuging the elute at 700,000 g for 16 h at 4°C and suspending the pellet in 1 ml of PBS; and vii. Quantifying the cfChPs contained in PBS in terms of their DNA content using a Pico-Green quantification assay. TMC01

[0064] In an embodiment, the kit-based method of Cell-free chromatin isolation comprises the steps of: i. Isolation of cfChPs from the serum using the ChromaFlash™ Chromatin Extraction Kit (Epigen tek) with some modifications. ii. Ultra-centrifugation of eeM-lml of serum at 700,000 g for 16 hours at 4°C to remove cellular debris, iii. Treatment of the pellet obtained with lysis bufferand protease inhibitor cocktail, followed by centrifugation at 700,000 g for 16 hours at 4°C and separation of the supernatant. iv. Suspending the pellet obtained containing cfChPs after removal of supernatant in the extraction buffer (extraction buffer and protease inhibitor cocktail), vortexed, and sonicated to enhance extraction. v. Sonication for ~20 seconds (twice). vi. Centrifugations at 8000 g for 10 minutes at 4°Cto remove residual debris and separation of the supernatant. vii. Addition of an equal volume of chromatin buffer to the supernatant and viii. Quantifying the cfChPs contained in chromatin buffer in terms of their DNA content using a Pico-Green quantification assay.

[0065] In an embodiment, the iPSCs are characterised by having: increase in the activation of OCT4, SOX2, KLF4 and NANOG genes as estimated by qRT-PCR, and expression of protein gene products as estimated by immunofluorescence. increased expression of stem cell surface markers selected from CD34, CD44, and CD 133 as estimated by flow cytometry.

[0066] • Formation of spheroids— the cfChPs treated cells in MammoCult Human Medium Kit for Mammospheres and Tumourspheres; and

[0067] • Teratoma formation following subcutaneous inoculation into SCID mice having three germ layers characterised by GFAP (ectodermal marker), TMC01

[0068] CD45 (mesodermal marker), and AFP (endodermal marker) as detected by immunohistochemistry.

[0069] The second of the present disclosure is to provide a kit for obtaining iPSCs.

[0070] In an embodiment, the kit comprises: a. cfChPs isolated from sera of healthy volunteers; b. somatic cell lines having capability to induce transcriptional activation Oct4, Nanog, KLF4 and Sox 2 in the chromatin particles; and c. Appropriate induction medium to induce pluripotency in somatic cells.

[0071] In an embodiment, the somatic cell lines are selected from NIH3T3 mouse fibroblast cells and human keratinocytes.

[0072] This invention marks a pivotal paradigm shift in stem cell production. By employing cfChPs mediated reprogramming, the invention effectively circumvents the intricacies and limitations inherent in traditional methodologies described above. The iPSCs generated through this innovative approach hold substantial promise, offering a scalable and consistent source of stem cells customised for specific therapeutic applications. This pioneering avenue not only addresses challenges related to cell yield and maintenance but also presents a potential solution to the ethical concerns associated with embryonic stem cells.

[0073] In conclusion, the innovative utilization of cfChPs for reprogramming somatic cells into iPSCs represents a transformative leap in stem cell production. This approach has the potential to revolutionize regenerative medicine, disease modeling, and drug discovery, paving the way for a new era of personalized and effective therapies.

[0074] The present disclosure is further described in light of the following experiments, which are set forth for illustration purposes only and not to be construed as limiting the scope of TMC01 the disclosure. The oligonucleotide pool mentioned here is only indicative in nature and can range from tens to hundreds of thousands or more. The following experiments can be scaled up to industrial / commercial scale and the results obtained can be extrapolated to industrial scale.

[0075] EXPERIMENT:

[0076] The NIH3T3 cells were treated with 10 ng of cfChPs isolated bya previously published standard method (described under example 1), while human keratinocytes were treated with 250 pg of cfChPs isolated by ChromaFlash™ Chromatin Extraction Kit (Epigentek) with some modifications (described under example 3).

[0077] Example 1: Isolation of cfChPs by the standard method:

[0078] In the process of isolating circulating cfChPs, blood samples were collected from patients with advanced-stage cancers as well as from healthy volunteers after obtaining informed consent. The blood processing procedure focused on serum isolation, which is the source for cfChPs. One ml serum ultra-centrifuged at 700,000 g for 16 hours at 4°C to remove cellular debris. The pellet obtained was treated with lysis buffer followed by ultracentrifugation at 700,000 g for 16 hours at 4°C and separation of thesupernatant and pellet obtained containing cfChPs re-suspended in PBS. cfChPs captured by passing the suspension through an affinity column containing a mixture of biotinylated anti-histone antibodies (20 pg each of Hl, H2A, H2B, H3, H4 in a volume of 1 mL) bound to Pierce® Streptavidin Plus Ultralink® Resin (Thermo Scientific, USA) (1 mL). The bound cfChPs eluted from the column using 0.25 M NaCl buffer and ultra-centrifuged at 700,000 g for 16 h at 4°C. Pellet re-suspended in 1 ml of PBS. Isolated cfChPs were quantified in terms of their DNA content using a Pico-Green quantification assay.

[0079] Example 2: Treatment of NIH3T3 cells with cfChPs from example 1

[0080] The experimental design had three groups: i. NIH3T3 untreated control cells TMC01 ii. NIH3T3 + cfChPs isolated from sera of healthy individuals (H-cfChPs): Treatment of NIH3T3 cells for 96 h with lOng of H-cfChPs, followed by serial passaging of the cells and harvesting them at passage numbers P2, PIO, P25, P50, and Pl 00. iii. NIH3T3 + cfChPs isolated from sera of patients with cancer (C-cfChPs): Treatment of NIH3T3 cells for 96 h with lOng of C-cfChPs, followed by serial passaging of the cells and harvesting them at passage numbers P2, PIO, P25, P50, and Pl 00.

[0081] A. Gene expression of stem cell-related transcription factors

[0082] The expression of key stem cell transcription factors (OCT4, NANOG, KLF4, and SOX2) was assessed in NIH3T3 cells treated with cfChPs using quantitative reverse transcription polymerase chain reaction (qRT-PCR). RNA was extracted at specific passage intervals — P2, P10, P25, P50, and P100 — following treatment with either H- cfChPs (from healthy donors) or C-cfChPs (from cancer patients). Untreated NIH3T3 cells served as the control. The housekeeping gene GAPDH was used for normalization. Relative mRNA expression levels were calculated and compared across passages to evaluate the persistence or decline of stem cell-associated gene expression in response to each treatment.

[0083] B. Flow Cytometry analysis of surface stem cell markers

[0084] Following the upregulation of stem cell transcription factor, its effect on phenotype was analyzed by checking the level of surface stem cell markers. The harvested cells from different passages were subjected to a thorough washing procedure involving two washes with IX phosphate -buffered saline (PBS) to ensure optimal cleanliness.

[0085] The cells were then transferred into a 5 ml flow tube, where specific antibodies targeting surface stem cell markers, namely CD44, CD34, and CD133, were introduced. The TMC01 antibody-cell mixture was incubated for a period of 20 minutes at room temperature, maintaining a light-protected environment to prevent photobleaching.

[0086] After the 20-minute incubation, the cells were washed once more with IX PBS to remove any excess or unbound antibodies. The cells were resuspended in 250 pl of PBS and acquired using Attune NxT flow cytometer (Thermo Fisher Scientific).The results obtained were analyzed via Flowjo software to gain insights into the surface expression of stem cell markers.

[0087] C. Spheroid Formation assay:

[0088] To assess spheroid formation, the following procedure was employed:

[0089] The experiment began by preparing MammoCult basal medium supplemented with proliferation supplement as per the manufacturer's instructions. This medium was then distributed into Ultra-low attachment (ULA) 96-well round-bottomed plates, with 100 pL added to each well using a multichannel pipette.

[0090] NIH3T3 control cells and cells treated with H-cfChPs and C-cfChPs were harvested and suspended in 1 ml of the prepared MammoCult basal medium. Following this, 1000 cells of each type were seeded into individual wells of the ULA plates.

[0091] The plates were placed in a controlled environment at 37 °C with 5% CO2 and left to incubate. On the seventh day, spheroid formation was assessed by capturing images of the wells using an Olympus 1X70 Microscope at 20X magnification.

[0092] Spheroids exceeding 60 micrometers in diameter were manually measured using ImageJ software to determine the mean spheroid diameter per well. The number of spheroids was counted visually. These processes allowed for the evaluation of spheroid formation and growth in response to cfChPs treatment.

[0093] The statistical test result highlights the clear impact of the different cfChPs treatments on spheroid formation in treated groups compared to the control group, emphasizing the profound effect of cfChPs derived from distinct sources on the behavior of NIH 3T3 cells. TMC01

[0094] D. Teratoma Formation assay and Immunohistochemistry (IHC):

[0095] We used an in vivo teratoma formation assay to test the capacity of the cfChPs-treated NIH3T3 cells to differentiate into the three germ layers. Teratomas are tumors that contain a variety of differentiated cell types, and their formation is a common test for evaluating the pluripotency of stem cells. One million cells from each NIH3T3 control, NIH3T3 + H-cfChPs and NIH3T3 + C-cfChPs group were inoculated subcutaneously into dorsal flanks of SCID mice. Teratoma growth was determined by palpation and mice were euthanized at ~ 8 weeks post-injection. Notably, all mice injected with cells from the NIH3T3 treated with C-cfChPs and H-cfChPsexhibited teratoma formation, while no tumors were observed in the NIH3T3 control group. This high level of efficiency in teratoma formation is a significant achievement, as it highlights the reliability and effectiveness of the approach.

[0096] Teratomas were fixed and embedded in paraffin. The sections were used for immunohistochemistry to detect the expression of GFAP (neural ectoderm) and CD45 (mesoderm) in an upright microscope at 20X.

[0097] In summary, the experiment confirmed the pluripotency of both H-cfChPs and C-cfChPs treated cells by demonstrating their ability to differentiate into both neuronal ectoderm and mesoderm cell types within the teratoma. The absence of marker expression in the negative controls and the positive expression in the appropriate controls validated the IHC results, supporting the conclusion that the injected cells possess pluripotent differentiation potential.

[0098] Example 3: Isolation of cfChPs using a commercially available kit:

[0099] In the process of isolating circulating cfChPs, blood samples were collected from healthy volunteers after obtaining informed consent. cfChPs were isolated from the serum using the ChromaFlash™ Chromatin Extraction Kit (Epigentek) with some modifications. The serum sample was first subjected to ultracentrifugation at 700,000 g for 16 hours at 4°C to remove cells and large debris, followed by a second ultracentrifugation at 700,000 gfor TMC01

[0100] 16 hours at 4°C with lysis buffer (lysis buffer and protease inhibitor cocktail) to further purify the chromatin. The resulting chromatin pellet was resuspended in extraction buffer (extraction buffer and protease inhibitor cocktail), vortexed, and sonicated to enhance extraction. The sample was then centrifuged to remove residual debris, and the supernatant was separated. An equal volume of chromatin buffer was added to the supernatant, and the isolated cfChPs were stored at -80°C. cfChPs were quantified in terms of their DNA content using a Pico-Green quantification assay.

[0101] Example 4: Induction of pluripotency in human keratinocytes by cfChPs from Example 3

[0102] Following the successful demonstration in murine cells, the approach was translated to human primary keratinocytes to confirm relevance in human somatic cells. Keratinocytes were treated with 250pg of cfChPs, a concentration determined through a dose-response viability assay.

[0103] Cell culture conditions:

[0104] Normal human epidermal keratinocytes (NHEK) of skin origin (passage 5) were obtained from Invitrogen. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) medium andl% antibiotic- antimycotic solution and cultured at 37 °C in a humidified atmosphere of 5% CO2.

[0105] Treatment of Human Keratinocyte cells with cfChPs:

[0106] Induction of iPSCs in human keratinocytes

[0107] The experimental design had two groups: i. Human keratinocyte untreated control cells ii. Human Keratinocytes + cfChPs isolated from sera of healthy individuals (H- cfChPs): Treatment of human keratinocytes for 72 h with 250 pg of H- cfChPs, followed by harvesting them.

[0108] A. Expression of stem cell-related transcription factors mRNA evaluated -PCR TMC01 qRT-PCR analysis was performed to assess the temporal expression of the stem cell- related transcription factors OCT4, NANOG, and KLF4 at multiple time intervals (6 h, 24 h, 48 h, 72 h, and 96 h). GAPDH was used as the control for normalization. Marked activation of all three markers was seen in cfChPs treated keratinocytes compared to the control keratinocytes.

[0109] Protein expression evaluated by immunofluorescence

[0110] To confirm the activation and localisation of sternness-associated transcription factors at the protein level, immunofluorescence staining was conducted on human keratinocytes treated with 250 pg of cfChPs for 72 hours. Untreated cells served as controls.

[0111] Following fixation with 4% paraformaldehyde and permeabilization with 0.1% Triton X- 100, cells were blocked with 3% BSA and incubated with primary antibodies against SOX2 (1:500), OCT4 (1: 100), and NANOG (1:500). Secondary antibodies conjugated to fluorophores were applied, and nuclear staining was performed using DAPI. Fluorescence microscopy revealed significantly increased nuclear localization and signal intensity of all three markers in treated cells compared to controls. These results confirm that cfChPs treatment activates a pluripotency-like transcriptional program in human keratinocytes.

[0112] All samples were imaged under identical settings to ensure quantitative comparison, and the experiment was repeated in duplicates for consistency.

[0113] B. Expression of surface stem cell markers

[0114] Flow cytometry assay was performed to assess the expression of surface sternness markers - CD34, CD44, and CD133, in human keratinocytes treated with cfChPs over a time course ranging from 6 to 96 hours post-treatment. cfChPs treatment led to an increase in expression of all three-stem cell-related surface markers compared to control cells. TMC01

[0115] C. Spheroid Formation assay

[0116] A spheroid formation assay was performed using cfChPs -treated and control human keratinocytes. The treated cells exhibited the ability to form compact, well-rounded spheroids, in contrast to the irregular and loosely aggregated structures observed in the control group (p<0.0001). This morphological difference was quantitatively validated by calculating the roundness scores using ImageJ software.

[0117] D. Teratoma Formation assay and Immunohistochemistry (IHC)

[0118] To evaluate the differentiation potential of cfChPs -treated cells, an in vivo teratoma formation assay was performed. One million control keratinocytes and cfChPs treated keratinocytes were inoculated subcutaneously into SCID mice (n=5 each group). In both groups, tumors developed in the animals (2 / 5 in the control keratinocytes inoculated mice and 2 / 5 in the cfChPs-treated keratinocytes inoculated mice) by 6 weeks. The tumours were harvested, fixed in formalin, embedded in paraffin, and sectioned for histological analysis.

[0119] Hematoxylin and Eosin (H&E) staining revealed that, unlike tumors induced by control keratinocytes, cfChPs-treated keratinocytes induced tumorshadstructures corresponding to derivatives of all three germ layers - ectoderm, mesoderm, and endoderm. These observations confirm the pluripotent-like differentiation capacity of cfChPs-treated cells in vivo. To further validate lineage-specific differentiation within the teratomas, immunohistochemistry (IHC) was performed using antibodies specific to germ layer markers. Paraffin-embedded teratoma sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Endogenous peroxidase activity was blocked, and sections were incubated with primary antibodies specific to: GFAP (ectoderm), and CD45 (mesoderm). Secondary antibody detection was performed using HRP -based systems and DAB as the chromogen, followed by hematoxylin counterstaining. Tumors induced by cfChPs-treated keratinocytes expressed markers of both ectoderm and mesoderm, while the control keratinocytes induced tumors, we negative for both. This substantiates the pluripotent-like properties induced by cfChPs in human somatic cells. TMC01

[0120] Results and summary: Treatment of somatic cells, namely, NIH3T3 and human keratinocytes with cfChPs resulted in the upregulation of core pluripotency-associated transcription factors suggesting activation of a pluripotent-like transcriptional program. Flow cytometry further demonstrated increased expression of stem cell surface markers CD34, CD44, and CD 133 in cfChPs-treated cells compared to controls confirming acquisition of sternness-associated phenotypes. Functionally, cfChPs-treated cells exhibited enhanced morphogenic behaviour, as evidenced by the formation of compact, spherical aggregates in spheroid formation assays indicating pluripotent nature of the cfChPs treated cells. cfChPs treated NIH3T3 and keratinocytes successfully formed teratomas containing histological structures representative of all three germ layers — ectoderm, mesoderm, and endoderm — as confirmed by hematoxylin and eosin (H&E) staining. Immunohistochemistry of paraffin-embedded sections verified the expression of different lineage-specific markers findings conclusively demonstrate that cfChPs treatment reprograms somatic cells toward a pluripotent-like state, enabling both transcriptional and functional hallmarks of pluripotency, including in vivo trilineage differentiation.

[0121] Key findings: The successful development of iPSCs without relying on retroviruses eliminates associated cancer risks, thus addressing one of the original objectives of the invention. Moreover, this novel approach has significantly boosted the reprogramming efficiency of somatic cells into iPSCs, fulfilling another key objective. Additionally, using cfChPs has the potential to make iPSC production more economically viable and rapid, aligning with yet another objective of the invention. Collectively, these findings strongly support the conclusion that, after the treatment with cfChPs, somatic cells underwent a remarkable transformation into cells with stem cell characteristics.

[0122] Moreover, these outcomes provide valuable insights that extend beyond the specific cell type used in this study. The implications of this invention can potentially reshape the field of stem cell biology. They can be applied to other cell types, offering a promising avenue for developing novel therapeutic strategies and regenerative medicine applications. This pioneering research sets the stage for further investigations to uncover the precise mechanisms and explore the broader implications of this transformative phenomenon.

Claims

WE CLAIM1. A method for obtaining Induced pluripotent stem cells (iPSCs) comprising the steps of: i. isolating cell-free chromatin particles (cfChPs) from human serum samples; and ii. treating the somatic cells selected from NIH3T3 mouse fibroblast cells and human keratinocytes, with the cfChPs to induce pluripotency, followed by characterisation of the iPSCs.

2. The method for obtaining induced pluripotent stem cells (iPSCs) as claimed in claim 1, wherein the induction medium activates pluripotency genes, selected from OCT4, SOX2, KLF4 and NANOG.

3. The method of obtaining Induced pluripotent stem cells (iPSCs) as claimed in claim 1, wherein the induction medium is Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% bovine calf serum (BCS), wherein the somatic cells are the NIH3T3 cells; and the induction medium is Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with foetal bovine serum (FBS) wherein the somatic cells are human keratinocytes; additionally, 1% antibiotic and anti-mycotic solution.

4. The method of obtaining Induced pluripotent stem cells (iPSCs) as claimed in claim 1, wherein the cells are cultured in a controlled environment in an incubator at 37°C with 5% CO2.

5. The method of obtaining Induced pluripotent stem cells (iPSCs) as claimed in claim 1, wherein the NIH3T3 cells is treated with 10 ng of cfChPs; and human keratinocytes is treated with 250 pg of cfChPs.

6. A kit for obtaining Induced pluripotent stem cells (iPSCs) comprising: a. cfChPs isolated from sera of healthy volunteers;b. somatic cell lines having capability to induce transcriptional activation Oct4, Nanog, KLF4 and Sox 2 in the chromatin particles; and c. appropriate induction medium to induce pluripotency in somatic cells, wherein the somatic cell lines are selected from NIH3T3 mouse fibroblast cells and human keratinocytes.

7. The Induced pluripotent stem cells (iPSCs) are characterized by having:increase in the activation of OCT4, SOX2, KLF4 and NANOG genes as estimated by qRT-PCR, and expression of protein gene products as estimated by immunofluorescence;'F increased expression of stem cell surface markers selected from CD34, CD44, and CD133 as estimated by flow cytometry;• Formation of spheroids by the cfChPs treated cells in MammoCult Human Medium Kit for Mammospheres and Tumourspheres; and• Teratoma formation following subcutaneous inoculation into SCID mice having three germ layers characterised by GFAP (ectodermal marker), CD45 (mesodermal marker), and AFP (endodermal marker) as detected by immunohistochemistry.

8. Use of Induced pluripotent stem cells (iPSCs) as claimed in claim 1, 7 and 7 in cancer research and therapy, offering powerful tools for disease modelling, drug discovery, immunotherapy, and tissue regeneration.

9. The method of obtaining Induced pluripotent stem cells (iPSCs) as claimed in claim 1, wherein the Standard method of cfChPs isolation comprises the steps of: i. Ultra-centrifugation of 1ml of serum at 700,000 g for 16 hours at 4°C to remove cellular debris, ii. Treatment of the pellet obtained with lysis buffer followed by ultracentrifugation at 700,000 g for 16 hours at 4°C and separation of the supernatant;TMC01 iii. Suspending the pellet obtained containing cfChPs after removal of supernatant; iv. Capturing the cfChPs by passing the suspension through an affinity column containing a mixture of biotinylated anti-histone antibodies (20 pg each of Hl, H2A, H2B, H3, H4 in a volume of 1 mL) bound to Pierce® Streptavidin Plus Ultralink® Resin (Thermo Scientific, USA) (1 mL) v. Eluting bound cfChPs from the column using 0.25 M NaCl buffer vi. Ultra-centrifuging the elute at 700,000 g for 16 h at 4°C and suspending the pellet in 1 ml of PBS; and vii. Quantifying the cfChPs contained in PBS in terms of their DNA content using a Pico-Green quantification assay.

10. The method of obtaining Induced pluripotent stem cells (iPSCs) as claimed in claim 1, wherein a kit-based method of cfChPs isolation comprises the steps of: i. Isolation of cfChPs from the serum using the ChromaFlash™ Chromatin Extraction Kit (Epigen tek) with some modifications. ii. Ultra-centrifugation of 1ml of serum at 700,000 g for 16 hours at 4°C to remove cellular debris, iii. Treatment of the pellet obtained with lysis buffer and protease inhibitor cocktail, followed by centrifugation at 700,000 g for 16 hours at 4°C and separation of the supernatant; iv. Suspending the pellet obtained containing cfChPs after removal of supernatant in the extraction buffer (extraction buffer and protease inhibitor cocktail), vortexed, and sonicated to enhance extraction. v. Sonication for ~20 seconds (twice). vi. Centrifugations at 8000 g for 10 minutes at 4°Cto remove residual debris and separation of the supernatant. vii. Addition of an equal volume of chromatin buffer to the supernatant; and viii. Quantifying the cfChPs contained in chromatin buffer in terms of their DNA content using a Pico-Green quantification assay.

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