DAIRY BIOTECHNOLOGY

Module 4. Cell culture and fusion technology

Lesson 18
STEM CELLS AND NUCLEAR CLONING

18.1 Introduction

Stem cell research is an emerging field with lot of potential for therapeutic applications in the management of untreatable diseases through cellular or tissue engineering.. Stems cells are useful both in basic as well as translational research. Looking into the tremendous potential of stem cell research, "CMC-DBT Centre for Stem Cell Research" has been established at CMC, Vellore to carry out basic and translational research. Facilities for carrying out research in the area of stem cells have been created at number of centres like PGIMER, Chandigarh; AIIMS, New Delhi; SGPGIMS, Lucknow, KEM hospital, Mumbai and LVPEI, Hyderabad. In the field of animal sciences, facilities have been created at Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001.

18.2 Definition of Stem Cells

Stem cells are the cells that regenerate and turn into cells that form tissues, organs and systems. The term ‘Stem’ comes from the word “Root’ which means ‘source’ i.e. stem cells are the source of all cells. The function of the stem cell is not known, however every single cell stems from this cell. Stem cells are undifferentiated cells having a high capacity for self renewal. They have the potential to become different types of cells. When stem cells divide, each new cell has the option either to remain as a stem cell or become a specialized cell with a particular function like muscle cell, blood cell nerve cell etc. Stem cells divide on receiving a signal. On receiving the signal, the genes are activated and stem cells start differentiating into a particular cell type (Fig. 18.1)

Stem cells are categorized into two types:

i) Embryonic stem cells

ii) Adult stem cells

18.2.1 Embryonic stem cells

Embryonic stem cells are isolated from the inner cell mass of blastocysts. In a developing embryo, stem cell can differentiate into all of the specialized embryonic tissues as they are totipotent (Fig. 18.2).

18.2.2 Adult stem cells

Adult cells act as a repair system and replenishes the worn out specialized cells and maintain turnover of regenerative organs like blood, skin and intestinal tissues.

18.3 Some Definitions Related to Stem Cells

18.3.1 Totipotent

Totipotent stem cells are those that can become any kind of cell in the body. ‘Toti’ originates from a Latin word which means whole or complete or total. After the fertilization of an egg, it undergoes a series of divisions to become an embryo before turning into a fetus. The cells that are formed during the first few divisions are ‘totipotent’. After 3 - 4 divisions of ‘totipotent’ cells, these cells will not be able to differentiate into any cell type.

18.3.2 Pluripotent

Pluripotent stem cells are those that have the ability to become almost any kind of cell in the body. Pluripotent stem cells result from totipotent stem cells after they have undergone first few divisions. Embryonic stem cells at the blastocyst stage and fetal stem cells are pluripotent. Both toti and pluripotent cells are essential for the development of new organism, hence they are found in the early stages of development in the embryo.

18.3.3 Multipotent

These stem cells can differentiate into a number of cells, but only those of a closely related family of cells. They are limited to mostly cells of the blood, heart, muscle and nerves. These cells function as a repair system for damaged tissues. The example in this category are Adult stem cells.

18.3.4 Unipotent

These stem cells can produce only one cell type and have the property of self renewal.

18.4 Applications

1. Stem cell therapy can be used for treatment of several diseases like Parkinson, cancer, myocardial infarction, heart muscle cells or insulin-producing pancreatic cells, missing teeth, crohn’s disease, leukaemia etc. (Fig. 18.3). Till date, bone marrow transplantation has been successfully achieved.

2. Stem cells are used in regenerative medicine for replacement of several cells and tissues as these can be directed to develop into heart muscle cells or insulin producing cells.

3. Stem cells can be genetically engineered to accomplish activities that they would not normally be programmed to do. This approach can be used to deliver the chemotherapeutic agents for treatment of cancers and tumors.

18.5 Stem Cell Research in India

India is one of the few countries in the world actually pursuing stem cell research as at least 20 research organizations and 15 companies such as Reliance Life Science and Lifecell are working on stem cells. Reliance Life, the pioneer in stem cell-based research in India on a commercial scale, has commercialized two products i.e. ReliNethra, a first-of-its-kind treatment in India for corneal blindness and ReliHeal-G, which quickly heals wounds. The company has completed clinical trials for treatment for heart attack using stem cells from the bone marrow of the patient. It has also been involved in carrying out clinical trials for application of stem cell-based therapies for skin disorders like stable vitiliago, non-healing diabetic ulcers, Parkinson’s disease and spinal cord injury.

LifeCell is involved in developing stem cell-based cardio vascular therapies in India. LifeCell is India's first private stem cell bank, where one can store the umbilical cord of the child for a fee, for future use. LifeCell has the largest network in India. The company has a network of over 50 centers in India and abroad. LifeCell started its operations in 2004 with cryogenic preservation of umbilical cord blood stem cells at its advanced facility in Chennai.

Stempeutics, funded by the Manipal Education and Medical Group, is planning to commercialize two drugs i.e. one for heart complications and the other for limb complications by 2011.

18.6 Stem Cell Therapy Centres in India

1. Tata Memorial - Mumbai
2. Adyar Cancer Center, Apollo Specialty Center- Chennai
3. Apollo Hospital, Global Hospital, NIMS - Hyderabad
4. Narayana Hruduyalaya, Manipal Hospital, Trinity Hospital -Bangalore
5. Christian Medical College - Vellore
6. R & R Army Hospital, AIIMS - New Delhi
7. Inlaks Hospital, Armed Forces Medical College - Pune
8. Sanjay Gandhi PGIMS - Lucknow
9. TRICELL - Chennai (A unit of Life Cell International Pvt. Ltd.)

18.7 Nuclear Cloning

Before learning nuclear cloning, let us be familiar with some terms related to the topic which will be used in this chapter.

18.7.1 What is a clone?

A clone is an exact genetic copy of a plant or animal or human being. It is defined as a group of genetically identical individuals descended from the same parent by asexual reproduction or group of genetically identical cells produced by mitotic division from an original cell.

The term ‘clone’ is derived from the Greek word ‘κλῶνος’, which means "trunk, branch", referring to the process whereby a new plant can be created from a twig.

18.7.2 What is a differentiated cell?

Multi-cellular organisms possess million of cells which function differently and become specialized. All cells originate from one unspecialized cell (undifferentiated) which then divides and become specialized cell by “Cell Differentiation” (Fig. 18.4). Differentiated animal cells can be induced to “start over” again and produce generic undifferentiated cells to develop a new organism. Dr. Wilmut succeeded by “reprogramming” a differentiated cell obtained from udder of an adult sheep.

18.7.3 Quiescence

Quiescence is the state of a cell when it is not dividing i.e. basic function of the cell is stopped. Quiescent cells cycling process is ‘arrested’ i.e. they stop dividing. Cell is not dormant or inactive e.g. Brain cells (neurons) – quiescent, yet busy in sending signals (Fig. 18.5).

18.7.4 Nuclear cloning or somatic cell cloning

Nuclear cloning also called somatic cell cloning is a technique in which the nucleus of a somatic cell is transferred into an enucleated oocyte for the generation of a new individual, genetically identical to the somatic cell donor. Various steps involved in nuclear cloning are shown in Fig. 18.6.

The steps involved in nuclear cloning are given below in a sequential order:

1. Cells are collected from a donor animal and cultured in vitro

2. A mature oocyte is then enucleated and a donor cell is transferred into the enucleated oocyte

3. The somatic cell and the oocyte is then fused

4. Embryo is allowed to develop into a blastocyst in vitro

5. The blastocyst can then be transferred to the recipient animal.

6. Cloned animals are born after completion of gestation

18.7.4.1 Dolly, the first cloned Sheep

Dolly, a Finn-Dorset Ewe, was the first mammal to be cloned by Ian Wilmut, Keith Campbell and colleagues at the Roslin Institute near Edinburgh in Scotland by nuclear cloning technique.. Dolly was born on 5^th July, 1996 and died at the age of six due to arthritis and lung disease. The cells used as donor were taken from the mammary gland. The success of cloning an entire animal, Dolly, from differentiated adult mammary epithelial cell created a revolution in science. This demonstrated that the genetic material from a specific adult cell, programmed to express only a distinct subset of its genes, can be reprogrammed to grow an entirely new organism. Later, this technique was successfully performed on several species including cat, rat, dog, monkey etc. and also for transgenic animals. Polly and Molly (Born 1997 - 2000), two ewes, were the first transgenic mammals to have been successfully cloned from an adult somatic cell. However, the difference between Dolly was that the nucleus that was transferred was from mammary gland whereas in Polly and Molly, it was from Fibroblast. Polly sheep clone produced Factor IX blood clotting factor.

18.7.4.2 Cloned Buffalo

Samrupa was the first cloned buffalo born on February 6, 2009, at National Diary Research Institute, India. However, it died five days later due to lung infection. Later, National Dairy Research Institute (NDRI) succeeded once again in producing a cloned buffalo calf Garima I (Fig. 18.7a and 18.7b) on 6^th June, 2010 and Garima-II weighing 32 kg, born through caesarian operation through the new and advanced ‘Hand-guided Cloning Technique', at NDRI, Karnal, on August 22, 2010. In the hand-guided cloning technique, the nucleus from the egg is taken by a hand-held fine knife so that the genetic material such as chromosomes, etc. come from single donor’s cell, that is, from one parent. Garima II is different from Garima I because, in this case the used donor cell was embryonic stem cell. In the previous cloning, the donor cell was from somatic cells. The donor embryonic stem cell was isolated from the eight-day old blastocyst. These cells were cultured up to 29 passages (117 days) till it expressed pluripotent marker and confirmed to be stem cell. The embryonic stem cells have better cloning ability as compared to somatic cells, as such the epigenetic reprogramming of these cells is much more efficient than the somatic cells, which are already differentiated and lineage committed.

Fig. 18.7 (a) Cloned buffalo calf Garima I at NDRI

Fig. 18.7 (b) One year old Garima I

National Dairy Research Institute, NDRI, Karnal also successfully cloned a male buffalo calf named ‘Shresth’ (Fig. 18.8) on 26^th August, 2010 through the new and advanced ‘Hand-guided Cloning Technique’.

Fig. 18.8 Shresth born at NDRI

Shresth weighing 41 kg was born through normal delivery with slight assistance carried out by doctors. This cloned buffalo calf is different from the earlier cloned calves as, in this case, the foster mother was provided opportunity for normal delivery. Shresth was produced from somatic cell from ear of 2 week old buffalo calf, and the embryo which led to successful pregnancy and normal delivery had remained frozen at -196^oC for one week in liquid nitrogen and brought back to active life upon thawing at room temperature. The earlier two calves were born through caesarean operation and were produced by using cells from fetus and embryonic stem cell, respectively.

The hand-guided cloning technique developed at NDRI, is an advanced modification of the “Conventional Cloning Technique”. The new technique is less demanding in terms of equipment, time and skill. Immature oocytes were isolated from ovaries and were matured in vitro. These were then denuded and treated with an enzyme to digest the outer layer of oocytes called ‘zona pellucida’. The oocytes were then treated with chemicals to push their genetic material to one side of the oocyte. This protruded side was then cut off with the help of “hand held fine blade” for removing the original genetic material of the oocyte. The enucleated oocyte was then electrofused with single cell taken from colony of embryonic stem cells. The resulting embryos were cultured and grown in the laboratory for seven days to develop them to the stage of blastocyst. The blastocysts were then transferred to recipient buffaloes.

This technology could go a long way in increasing the number of superior milch buffaloes in India. Moreover, this technology can also help by breeding bulls in the shortest possible time.

18.7.5 Applications of Nuclear Cloning

1. With the help of nuclear cloning, thousands of genetically identical copies of mammals can be produced.
2. High quality offsprings with specifically selected traits like increased milk production in cows etc. can be produced.
3. It can also be used to produce transgenic animals for pharmaceutical protein production or xeno-transplantation or to preserve endangered species.
4. Somatic cloning is an essential tool for studying gene function, genomic imprinting, genomic re-programming, regulation of development, genetic diseases, and gene therapy.

18.8 Disadvantages of nuclear cloning

1. Low survival rate or low efficiency - 62% fetal loss rate in cloned animals compared to 6% in naturally fertilized animals.
2. High incidence of developmental abnormalities - More than 60% of cloned animals die shortly after birth due to weaknesses and dysfunctional organs.
3. Costly and inefficient.
4. Can lead to a monocultural animal population i.e narrowing of gene pool and thereby, can cut a population’s genetic diversity to zero.

Internet Resources

http://en.wikipedia.org/wiki/Stem_cell

http://en.wikipedia.org/wiki/Stem_cell_treatments

stemcells.nih.gov/info/basics/

http://www.internationalstemcellservices.com/stem-cell-therapy.html

http://en.wikipedia.org/wiki/Somatic-cell_nuclear_transfer

http://en.wikipedia.org/wiki/Dolly_%28sheep%29

http://www.roslin.ed.ac.uk/public-interest/dolly-the-sheep/