Stem Cells

Stem Cells

The Stem Cell Reality


Stem cells and their applications have been making headlines with regularity, for the right as well as wrong reasons. Leading magazines such as National Geographic have carried whole issues devoted to stem cells. Certain terms used when talking about stem cells need to be explained.

Definitions


“Stem cell research”
 includes research into various aspects of stem cell biology, and in vivo and ex vivo manipulation etc of stem cells from various sources.
“Totipotent” refer to those stem cells in the 1-4 day old blastocyst, which are capable of differentiation into all the 230 odd cell types in the body.

“Pluripotent” cells were believed to be those cells harvested from the 5-7 day old blastocyst that can differentiate into most of the cell types and which has been used for the production of embryonic stem cell lines.

“Multipotent” are those stem cells that can differentiate into many other kinds of cell types

More recently researchers have been able to de-differentiate the pluripotent stem cell to become totipotent. The multipotent adult stem cell has also exhibited more pluripotency than thought possible.

Unipotent – those cells that belong to a specialized lineage and that are capable of self-replication without differentiation.

Sources of stem cells

  • Embryo 5-7 day old blastocyst
  • Non-embryonic or Adult stem cell sources, which are

1)  The adult bone marrow and peripheral blood
2)  Umbilical cord and placenta

Embryonic stem cells

Human ova that are fertilised through in vitro fertilization (IVF) and have been cryo-preserved are used for procuring stem cell lines for research or therapeutic purposes.  At the moment there are several such stem cell lines in circulation all over the world. In the process of finding one such perfect embryo, several other embryos are destroyed. Ethical issues and dilemmas surround the usage of these cell lines and most countries have formulated their own guidelines for usage.
Designer specific stem cell lines have been created by a process of Somatic Cell Nuclear Transfer (SCNT) using either cryo-preserved oocytes (unfertilized ova) or fresh oocytes.

A cryo preserved unfertilized ova or a fresh unfertilized ova is used. The DNA of this is removed and instead the DNA from a non-reproductive cell belonging to the person for whom the cell lines need to be prepared is inserted. This being a somatic adult cell has the full chromosomal complement and the unfertilized ovum in effect is now fertilized and a replica or clone of the individual from whom the DNA was taken. This is now allowed to grow to the 5-7 days blastocyst stage, when the stem cells are harvested and used for the preparation of a specific cell line tailor – made to one individual. This is also known as therapeutic cloning as opposed to reproductive cloning where the tailored embryo is re-implanted in a uterus to produce a “tailor made or genetic design specified” baby. Such designer specific babies include Dolly the cloned sheep and most recently Snuppy the cloned dog. Human cloning is now a distinct possibility and the implications of this being tremendous, most countries are not in favour of reproductive cloning.

Another contentious issue is that young healthy female volunteers have been lured with large amounts of money and subject themselves to extensive and repeated cycles of hormone therapy.

Adult stem cells

This “safe” source of stem cells is harvested mainly from the bone marrow (most frequent source) and the peripheral blood. Peripheral blood stem cell harvesting is fast replacing the bone marrow, as unlike bone marrow harvesting, it is a relatively simple process that does not require an anaesthetic. These are referred to as the “pluripotent” stem cell. The bone marrow stem cell can be mobilized into the peripheral blood by giving the donor granulocyte colony stimulating factor (CGSF) The donor is injected with CGSF a few days before the proposed harvest in a few sittings depending on the yield required and through a process of leucapheresis the WBCs are collected and then the tem cells are separated.
The third and equally non-controversial source of stem cells that is gaining ground in recent times is the umbilical cord and placenta. These cells are derived from the umbilical cord at the time of delivery, and until 1988, this was routinely discarded as biological waste. They have a few advantages over both other sources and hence researchers now view this as a better option.

Stem cells derived from the bone marrow and the umbilical cord were initially believed to be mainly the heamatopoietic stem cells (HSC) that mainly have heamatopoietic capabilities. HSC themselves have been found to be of two kinds: the long term stem cells that are capable of indefinite self renewal through the entire lifespan of the organism and the short term progenitor or precursor cells. They are capable of proliferating, but they have a limited capacity to differentiate into more than one cell type as HSCs do. The long-term replicating HSCs are most important for developing HSC-based cell therapies.

More recently scientists have identified the mesenchymal or stromal stem cell (MSC) from the bone marrow that is capable of differentiation into a variety of cells eg muscle, nerve, skin and so on. Mesenchymal stem cells have been studied in great detail and scientists have advanced knowledge about how to grow these cells in culture. As these cells can be obtained quantities sufficient for clinical applications, they are ideal candidates for use in tissue repair.

Comparison of stem cells from different sources

Properties Embryo Umbilical Cord Adult 
Quantity of yield Maximum yield Least yield Moderate
Method 5-7 day old embryo Umbilical vein after clamping of cord Needle inserted into flat bone
Impact Death of the embryo Entirely harmless Discomfort for a few days after procedure
Contamination Least Virtually Nil Likely
Availability Controversial Plentiful On Demand mainly
Search time Not applicable Already banked Search time double
HLA typing Required 3/6 or 4/6 sufficient 5/6 or 6/6 preferable
GVHR Least 38% Around 75%
Engraftment Not applicable Slow Good
Effectiveness Still mainly in research Very good Very good

Other sources of stem cells

  • Fat has been identified as one of the richest sources – 150 ccs of fat is supposed to contain 4 million cells
  • Olfactory Ensheathing Cells (OEC)
  • Amnion
  • Cord lining and Wharton’s Jelly
  • In addition almost every organ in the body has a small quantity of stem cells.
What makes stem cells unique
      1)   Varying degrees of immunological naiveté depending on the source of the cell
2)   Plasticity or the ability to differentiate
3)   Multiplication without differentiation indefinitely
4)   Ability to home-in to the affected area or target organ.

How are stem cells identified?
The challenge of identifying these stem cells is difficult for the following reasons:

1)  Just about 1 in every 10,000 to 15,000 bone marrow cells is thought to be a stem cell.
This proportion further  reduces to 1 in 100,000 blood cells in the peripheral blood.
2)  Second, there are multiple types of stem cells.
3)  The stem cells look similar to many other blood or bone marrow cells

In 1988 Weissman and his colleagues identified a set of protein markers on the mouse stem cell surface that were indicative of long term stem cells. Later human markers were similarly identified. These markers exist on the cell surface of undifferentiated cells in vivo and in vitro. As these cells begin to develop as distinct cell lineages the cell surface markers are no longer identified

Proposed cell-surface markers of undifferentiated heamatopoietic stem cells.
Mouse Human
CD34low/- CD 34+
SCA-1+ CD59+*
Thy1+/low Thy1+
CD38+ CD38low/-
C-kit+ C-kit-/low
lin* lin**
* Only one of a family of CD59 markers has thus far been evaluated.
** Lin cells lack 13 to 14 different mature blood-lineage markers.

Such cell markers are tagged with a monoclonal antibody labelled with a fluorescent dye and can be culled out from a mixture of cells. These however are a mixture of long term, short-term progenitor and non-stem cells.

Applications of stem cells

Heamatopoietic stem cells (HSC) have been used in a variety of conditions and their unique properties in addition to those given above are

      1)   They can be mobilized out of the bone marrow into the peripheral circulation.

2)   Can undergo programmed cell death or apoptosis, a process by which cells that are either detrimental
or not required can self-destruct.

Table I: Common Indications for Hematopoietic Transplantation1

A Malignant

Hematological
AML (1st remission; except APML)
ALL (2nd remission)
ALL (1st remission for high risk cases)
CML
CLL (young)
MDS
Non Hodgkin’s Lymphoma (relapsed / high risk)
Hodgkin’s disease (multiple relapsed)
Multiple Myeloma

Non-Hematological

Breast Cancer
Neuroblastoma
Testicular Cancer
Ovarian Cancer
Intracranial neoplasms
Lung Cancer
Malignant Melanoma
Renal Cell Carcinoma (metastatic)

B Non malignant genetic disease

Thalassaemia major
Severe combined immunodeficiency (SCID)
Sickle cell disease
Kostmann’s syndrome
Chronic granulomatous disease
Chediak-Higashi syndrome
Diamond-Blackfan syndrome
Congenital aplastic anemia
Fanconi’s anemia
Osteopetrosis
Hurler’s syndrome
Lysosomal storage disorders
Wiskott Aldrich Syndrome

C Non malignant acquired disease

Severe Aplastic Anemia

For a stem cell transplant from any source to be most effective, the disease needs to be in a state of remission. In cord blood stem cell transplants in addition the number of CD 34 cells per kilogram of body weight and the HLA matching are crucial to the success.2

Ongoing trials in various areas include

Cardiac diseases – Myocardial infarctions, Cardiac Failure, Cardiomyopathies etc
Neurological – Stroke, Parkinson’s disease, Muscular Dystrophy, Multiple Sclerosis
Diabetes
Spinal cord injuries using stem cells from various sources

Future therapeutic applications include

Alzheimer’s disease
Autoimmune disorders
HIV infection
Liver diseases

Recent areas of research

While stem cells from the bone marrow have been used extensively all over the world, more recently, researchers in Japan have shown that in patients suffering from Myelo Dysplastic Syndrome (MDS), the umbilical cord could be considered an equally effective and alternative source when allogeneic bone marrow matches were not available. 3

Researchers have identified in the last few years, specific enzymes that need to be inactivated if homing and engraftment of the transfused stem cells is to be improved. Cell expansion and double cord transplantation techniques have helped improve the number of stem cells for transplant even to large individuals. The heaviest individual who has benefited from a transfusion with stem cells from cord blood weighed 85 kilograms and with the above-mentioned techniques, this potential for usage of these stem cells is only going to increase. Using one of the culture techniques, recently Korean researches have successfully helped a 37-year-old woman who had been paralysed for 20 yrs following a spinal cord injury walk.4

Adults with leukemia have been given a peripheral blood stem cell transplant along with a cord blood transplant from the same source. Mini transplants of cord blood have been used effectively in the treatment of recurrent Mantle cell carcinoma, along with a less strong myelo-ablative regimen banking on the graft versus tumour effect to remove the remaining malignant cells.

Stem cells are playing an increasingly important role in the field of cosmetic surgery too. Wrinkles, signs of ageing and baldness have been treated successfully using either stem cells from the fat or from the hair bulb. They have been found to be more effective as breast implants more recently. Patients with burns have been treated effectively with skin stem cells.

Stem cells have been differentiated into islet cells capable of producing insulin and will be of great help to Type 1A diabetics. Infarcted areas of the brain have been treated effectively in mice using mannitol along with stem cells derived from the umbilical cord. According to researchers in the Medical College of Georgia and University of South Florida, it has been noticed in mice, that the infracted area has been found to reduce by as much as 40 % in the two days following transfusion of the cells. Similar research done by a team from Stanford University reiterates the findings.5

In myocardial infarcts too, researchers have identified that a heart to heart transfer of stem cells was able to regenerate more rapidly and effectively in infracted tissue improving the vascularity and contractility of the cardiac musculature.

What we still don’t know about stem cells

With each day, new avenues of treatment are opening up; potentially new cures are being identified while some treatments are being modified. Stem cell research and applications have increased tremendously and has opened up a field of Regenerative medicine that until a few years ago was considered impossible. To realize the promise of novel cell-based therapies for pervasive and debilitating diseases listed above and several more that researchers are working on, a lot more about basic stem cell physiology needs to be understood.

Scientists must be able to easily and reproducibly manipulate stem cells so that they possess the necessary characteristics for successful differentiation, transplantation and engraftment. To be useful for transplant purposes, stem cells must be reproducibly made to:

  • Proliferate extensively and generate sufficient quantities of tissue.
  • Differentiate into the desired cell type(s).
  • Survive in the recipient after transplant.
  • Integrate into the surrounding tissue after transplant.
  • Function appropriately for the duration of the recipient’s life.
  • Avoid harming the recipient in any way.

At this point in time, stem cells hold out immense promise as an answer to many of man’s hitherto untreatable illnesses. Man must effectively “control” stem cells and coax them into behaving in a predictable fashion repeatedly. That day is not far away and when this happens, stem cells can be considered the “magic potion”.

References

1)   HEMATOPOIETIC STEM CELLTRANSPLANTATION IN INDIA
Parikh Purvish M, Shah Pankaj M, Easow Jose Kumar Lalit

2)   Successful transplantation of HLA-matched and HLA-mismatched umbilical cord       blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease. Blood. 1996; 88:795-802. Wagner JE, Rosenthal J, Sweetman R, et al

3)   Heamatopoietic Stem-Cell Transplants Using Umbilical-Cord Blood. Volume   344:1860-1861 June 14, 2001 Number 24

4)   Stem cells make paralytic walk: Spine repaired using umbilical cord blood. –AFP

5)   Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. PNAS | August 10, 2004 | vol. 101 | no. 32 | 11839-11844. S. Kelly, T. M. Bliss, A. K. Shah, G. H. Sun, M. Ma, W. C. Foo, J. Masel, M. A. Yenari, I. L. Weissman, N. Uchida, T. Palmer and G. K. Steinberg.


Dr Saranya Nandakumar
Consultant Microbiologist
[email protected]

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