The Successful Reprogramming Of Somatic Cells Health And Social Care Essay

Submitted by

KARANVIR SINGH KANWAR

Enrollment No. A0510711059

In partial fulfilment for award of degree

Of

MASTER OF TECHNOLOGY

IN

BIOTECHNOLOGY

AMITY UNIVERSITY NOIDA CAMPUS

UTTARPRADESH

10 December – 29 April 2013

ACKNOWLEDGEMENT

I wish to place in record my deep sense of gratitude and profound thanks to my respected supervisor Mr. Subhanjan Bhowmik, MD Bencos Research Solution for his complete guidance and constant support during the course of the summer training project.

I would like to thank Dr. Mukesh (NII), Miss Pooja (AIIMS), and Mr. Rupesh(IISc) who provided us the MEF cell line, Embryonic stem cell line, and vectors respectively without which this project have not been possible.

I would like to express appreciation for the generous support provided by Mandeep Kaur, Scientific Officers, Bencos Research Solution. Their Kind Corporation and support helped me a lot to complete my project successfully.

I would also like to extend my gratitude to my guide, Dr. Nidhi Shrivastava, for guiding me through every step of the project.

I would also like to thank my senior and fellow trainee at Bencos, Ms Mehak Sharma for her constant support, cooperation and guide throughout the project.

Last but not the least I would like to thank my family and all my friends of Bencos Research Solution for their undying moral support and encouragement.

Place: Noida

Date: 29/04/2013

(KARANVIR SINGH KANWAR)

DECLARATION

I hereby declare that the summer training entitled "Oct 4 induced iPSC generation in HeLa cells" to AMITY INSTITUTE OF BIOTECHNOLOGY, AMITY UNIVERSITY, Uttar Pradesh, is a record of original work done by me under the guidance of Subhanjan Bhowmik, Bencos Research Solutions Pvt. Ltd., New Delhi and this project has not performed the basis for any award or degree or diploma/associateship/fellowship.

KARANVIR SINGH KANWAR

Enrollment No. A0510711059

CERTIFICATE

TABLE OF CONTENTS:

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Contents

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ABSTRACT:

The cancer stem cell (CSC) hypothesis proposes that CSCs are responsible for metastasis and disease recurrence. Therefore, targeting CSCs has the potential to significantly improve outcomes for cancer patients. The Oct4 transcription factor gene is a master gene that plays a key role in the self-renewal and pluripotency of stem cells. The Oct3/4 gene, a POU family transcription factor, has been noted as being specifically expressed in embryonic stem cells and in tumor cells but not in cells of differentiated tissues. With the ability to isolate adult human stem cells it became possible to test for the expression of Oct3/4 gene in adult stem cells and to test the stem cell theory of carcinogenesis. Using PCR primers for Oct4 in Embryonic stem cells, HeLa cell line, and MeF (mouse embryonic fibroblast) its presence in these three cell lines was validated. PCR results depicted that among these three cell lines embryonic stem cells showed the presence of Oct4. The Oct4 gene was then inserted into pET28a bacterial expression vector. Bacterial cells used for transfection were BL12. Presence of intact Oct4 protein was validated using SDS and sequencing of the insert into vector was done to check any mutation in gene. Oct4 gene can then be used to express stemness in HeLa cell line through inter-transfection. Whole experiment was done to check the role of Oct4 in providing stemness. This can be used to generate iPCSCs (induced pluripotent cancer stem cells) which can then be used to study the properties of cancer stem cells in vitro and, the role of Oct4 expression in providing stemness to these cells. Role of Oct4 expression in providing stemness will also prove useful target for stem cell-specific cancer therapy.

INTRODUCTION:

The cancer stem cell (CSC) hypothesis proposes that CSCs, which constitute a small population of cells within a tumor, are able to self-renew and to give rise to diverse cellular progeny. Indeed, CSCs may be responsible for the escape of tumors from conventional therapies, resulting in residual CSCs that can lead to disease recurrence and metastasis [1- 3]. Therefore, CSC-specific therapies might prevent cancer relapse and completely destroy cancer at its root.

The OCT4 (also known as POU5F1) transcription factor gene is known as a master pluripotency gene [4, 5]. Kim and colleagues showed that OCT4 alone is sufficient to directly reprogram adult mouse neural stem cells to pluripotent stem (iPS) cells [6]. Clinical studies have shown that tumors with intense expression of the OCT4 stem cell marker are associated with further disease progression, greater metastasis, and shorter cancer related survival compared with tumors with moderate and low OCT4 expression [7]. Furthermore, OCT4 is reported to be a core regulator of stem cell self-renewal and differentiation, and was recently validated as a CSC target [8].

Loss of Oct4 function early in development causes cells of the preimplantation embryo to acquire a trophectodermal fate (Nichols et al., 1998), whereas loss in the GC lineage leads to apoptosis of primordial germ cells (PGCs) (Kehler et al., 2004). These studies of the developing embryo were complemented by studies of ESCs in which the Oct4 function was abolished (Niwa et al., 2000). ESCs are especially interesting in this context, as they are derived from the inner cell mass of the blastocyst and thus represent a developmental stage following the establishment of the pluripotent founder population. While loss of Oct4 function results in differentiation into trophectodermal cells, overexpression of Oct4 in ESC leads to differentiation along the mesodermal and primitive endodermal lineages.

REVIEW OF LITERATURE:

The successful reprogramming of somatic cells to induced pluripotent stem (iPS) cells indicates that differentiated cells retain the capacity to revert to immature cells. Oct4, Sox2, c-Myc, Klf4, Nanog and Lin28 have been used for somatic cell reprogramming (Takahashi et al., 2007; Yu et al., 2009b). Oct4 is the most critical transcription factor since it can reprogram adult stem cells to iPS cells as a single factor (Kim et al., 2009a, b). Tumorigenesis and somatic cell reprogramming

share common mechanisms (Daley, 2008). Aberrant expression of Oct4, Nanog, Sox2, Lin28 and Klf4 is associated with abnormal tissue growth or tumorigenesis (Hochedlinger et al., 2005; Chen et al., 2008; Schoenhals et al., 2009; Viswanathan et al., 2009).

Tumor dedifferentiation is a well-known phenomenon and it has long been proposed to be involved in tumor progression (Gabbert et al., 1985). Similar to somatic cell reprogramming, tumor dedifferentiation is reversal of cell development to a more immature state. Dedifferentiated melanoma cells are known to lose pigmentation (Bennett, 1983). Cancer stem cells (CSCs) have dedifferentiated phenotypes and it has been shown that CD271þ melanoma CSCs lack expression of common melanocytic markers (Boiko et al., 2010). However, the mechanism underlying tumor dedifferentiation is not fully understood.

There is enormous interest to find the origin of CSCs and target these cells for therapy. Oct4 has been proposed as a biomarker for CSC-like cells. Oct4 is detectable in a variety of cancer types including melanoma (Strizzi et al., 2008) and CSC-like cells are enriched for Oct4 expression (Saigusa et al., 2009; Hu et al., 2010; Liu et al., 2010; Peng et al., 2010; Zhang et al., 2010). It has been shown that Oct4 expression is associated with differentiation state of cancer cells (Chen et al., 2009; Zhang et al., 2010).