Pluripotent and Multipotent Stem Cells

• What are Stem Cells?
• A Brief History of Stem Cell Research
• Types of Stem Cells
• Stem Cell Research Applications
• Autoimmune Disorders
• Stem Cell Research Challenges

What are Stem Cells?

Stem cells have the unique ability to self-renew or to differentiate into various cell types in response to appropriate signals within the body. These properties provide stem cells with unique capabilities for tissue repair, replacement, and regeneration. Because of these properties, stem cells have become valuable research tools for regenerative medicine and possible stem cell therapies.

The origin of stem cell research dates backs to 1960s with the discovery of hematopoietic stem cells within bone marrow by Drs. James Till and Ernest McCulloch1. A brief timeline of stem cell research can be seen below.

A Brief History of Stem Cell Research

1963

Discovery of renewing cells in bone marrow

1968

Bone marrow transplantation between two siblings

1981

Embryonic stem cells isolated from mouse blastocysts (Nobel prize)

1992

Adult stem cells identified in human brain

2001

Mouse embryonic stem cells created using nuclear transfer technique

2006

Discovery of induced pluripotent stem cells (Nobel prize)

2010

Medical treatment using human embryonic stem cells for spinal injury

2014

Embryonic stem cells from adult stem cells (Insulin producing Beta cells generated from skin cells)

Types of Stem Cells

Three important features of stem cells are the basis for their classification:

• Stem cells have unlimited self-renewal capabilities.
• Stem cells are non-differentiated cells with unspecialized functions
• Stem cells can differentiate into specific cell types under appropriate conditions.

Stem cells are broadly characterized into multipotent stem cells or pluripotent stem cells.

Multipotent Stem Cells

This category includes adult stem cells that demonstrate the ability to self-renew or to differentiate into specialized cell types present in a specific tissue or organ. Examples include hematopoietic stem cells that specialize into various blood cells, mesenchymal stem cells that can give rise to osteoblasts, myocytes, chondrocytes and adipocytes² and neural stem cells which differentiate into neurons, astrocytes, and oligodendrocytes.

Key features of multipotent stem cells:

• Present in small number in specialized tissues
• Primary function is to replenish damaged or apoptotic cells
• Remain quiescent until they perceive signals to differentiate into specific type of cells

Increasing number of studies are exploiting multipotent stem cells in tissue engineering, treatment of leukemia and skin grafting for burn victims and cosmetic enhancements.

Pluripotent Stem Cells

Pluripotent stem cells are often referred to as true stem cells since they have the ability to differentiate into any type of cell lineage. They are classified into embryonic stem cells, perinatal stem cells and induced pluripotent stem cells based on the tissue of origin.

Embryonic Stem Cells (ES Cells)

Embryonic stem cells are pluripotent stem cells derived from embryos; from the inner cell mass of blastocyst stage, approximately after the 5th day of fertilization. Cells harvested within first four cleavages of the embryo, they are considered totipotent. Totipotent cells have an additional ability to differentiate into extraembryonic tissues along with germ layer derived tissues. ESC has an ability to divide indefinitely in-vivo, unlike the adult stem cells which divide only during tissue damage or cell death. Although a rich source of stem cells with wide applications, the use of embryonic stem cells has ethical challenges. Historically, mouse embryonic stem cells have been a valuable tool to generate transgenic knock-out mouse models.

Perinatal Stem Cells (Umbilical Cord Stem Cells)

Perinatal stem cells are derived from umbilical cord blood and are most widely used pluripotent stem cells. The usage of perinatal stem cells has less ethical issues than the embryonic stem cells; it is because the tissues from which they are isolated are otherwise being discarded³. Cord blood banking is increasingly being accepted as an option to treat complicated disorders in the later part of an individual’s life. Perinatal stem cells isolated from one’s cord blood and used for transplant overcome HLA antigen incompatibility. The challenges include low cell counts that reduce the success of grafting.

Induced Pluripotent Stem Cells (iPSCs)

Induced pluripotent stem cells (iPSCs) are adult cells reprogrammed artificially to behave like embryonic stem cells. The advantage of induced pluripotent stem cells is the reduced chances of graft rejection since it uses somatic cells from the same individual. Two methods of induction are popular:

• Somatic cell nuclear transfer (SCNT): Nucleus from the fertilized zygote or unfertilized oocyte is replaced with the nucleus of a somatic cell of a donor⁴.
• Nuclear reprogramming: somatic cells are reprogrammed with the transfection of a specific set of transcription factors (Oct3/4, Sox2, Klf4 and c-Myc)⁵. The advanced nuclear reprogramming is more effective than SCNT and has important therapeutic implications⁶.

Table 1. Various stem cells types exist including totipotent, pluripotent and multipotent cells.

Stem Cell	Source	Function	Pros	Cons
Totipotent	4 cell embryo	Can differentiate into all cells (including extra embryonic cells)	Easy to isolate and grow Unlimited self renewal	High risk of tissue rejection Ethical issues Teratoma formation
Pluripotent	Blastocyst, Umbilical Cord, Reprogrammed iPSCs	Differentiate into cells derived from 3 germ layers	Easy to isolate and grow Unlimited self renewal	High risk of tissue rejection Ethical issues Teratoma formation
Multipotent	Organs (with specialized tissues)	Differentiate into specialized lineage of cells	Less likely of tissue rejection Less ethical issues	Hard to isolate Limited differentiation

Stem Cell Research Applications

The ability to self-renew and differentiate into mature cell types makes stem cells attractive targets of basic research. Most research is involved in the identification of differentiation factors, genetic as well as environmental signals that exist in the stem cell niche^8-9. Increasing number of clinical applications is being developed in the following areas:

Neurological Diseases

Amyotrophic Lateral Sclerosis (ALS)

ALS is progressive neuromuscular disease, characterized by loss of motor neurons in the brain and spinal cord. The treatment strategies only alleviate the symptoms. Studies aim to regenerate lost motor neurons in the brain and spinal cord with bone marrow stem cells and iPS. Experiments with prenatal stem cells and mesenchymal stem cells feature in the early phase of clinical trials¹⁸.

Spinal Cord Injuries

Severe accidents, falls, and birth defects like spina bifida cause serious injury to spinal cord. In several cases, nerve fiber bundles are severed, leading to paralysis. Currently, clinical investigations aim to use adult stem cells to regenerate new nerve cells and trigger growth of severed nerve fibers. Investigations to transplant support cells that wrap nerves with myelin sheath are also being carried out. In the absence of existing therapies, employing stem cells to improve nerve function is critical¹⁹.

Eye Diseases

Injuries to cornea, retina or optic nerve and disease conditions such as age-related macular degeneration, glaucoma and retinitis pigmentosa result in vision loss. Improved vision was noticed in animals with macular degeneration when transplanted with retinal pigment epithelial cells derived from embryonic stem cells limbal stem cells and retinal stem cells have shown positive results in regenerating human corneal tissue²⁰.

Wound Healing

Conventional skin grafts fail to restore the complete composition of dermis lost due to injuries, genetic disorders, and burns¹⁰. Basal layer stem cells derived skin grafts are approved for clinical use, which are mainly used for large burns. Stem cells promote better and faster healing of the burn wounds and decrease the inflammation levels with less fibrosis and scar progression¹¹.

Cardiovascular Diseases

Most cardiovascular disorders are characterized by ischemia and heart muscle injury that result in arrhythmias, hypertrophy and congestive heart failures. Standard treatments include surgeries repairing blocked arteries, medications reducing fluid retention and lifestyle changes. However, none of them can regenerate the heart tissue. Stem cell therapies, under investigation, aim to restore lost function of heart tissue and blood vessels¹² and reduce clinical cardiac events¹³.

Autoimmune Disorders

Type 1 Diabetes

Hyperactivity of body’s immune system destructs insulin-producing pancreatic beta cells in Type 1 diabetes patients. While insulin injections are effective treatment options, they fail to provide a consistent and right amount of insulin required throughout the day resulting in unstable levels of glucose in the blood. Cells derived from haematopoietic stem cells are being investigated to replenish beta cells. Another study is reprogramming sperm stem cells into embryonic-like cells, which further differentiate into beta cells of pancreas¹⁴.

Multiple Sclerosis

Multiple sclerosis is characterized by a chronic inflammatory diseases in brain or spinal cord triggered by body own immune system. Majorly, myelin sheath of the nerves is damaged which compromise nerve signals. There is no cure for multiple sclerosis; however, medications are used to treat symptoms. Bone marrow stem cells and neural stem cell derived oligodendrocytes are being investigated to regenerate neurons with a myelin sheath. In addition, investigations to reduce immune system function are also being carried out^15-16.

Arthritis

Arthritis is characterized by chronic pain, inflammation of the joints and stiffness. There are many etiological reasons; some of them include destruction of cartilage by the immune system. Current medications majorly reduce pain and inflammation. Stem cells have a potency to differentiate into chondrocytes, which makes cartilage. Investigations are underway to make patient-derived chondrocytes for transplantation¹⁷.

Disease Modeling & Drug Screening

Stem cells are evolving as suitable models to study the efficacy of drugs since they combat the shortcomings of transformed cell line systems and bypass the ethical challenges of using animal models²¹. Now pharmaceutical industries are increasingly focusing on stem cells for screening drugs²² and creating “disease-in-a-dish” cellular models. Some of the applications are as follows:     

• Human embryonic stem cells derived cardiomyocytes (hESC-CMs) provide human model for predictive cardiac toxicity, reducing drug development costs²³.
• Induced pluripotent stem cells derived from patients resemble pathogenic conditions in-vitro and could increase the success rate of drug screening and accelerate drug development process²⁴.
• Differentiated neuronal cells are similar in genetics and biological content to human brain cells compared to animal disease models. These neuronal cells derived from stem cells are employed for cell viability, calcium response and neurite outgrowth assays^25-26.

Stem Cell Research Challenges

Stem cells are an evolving area of research that is riddled with multiple unknowns.

Immunological Rejection

A major challenge with stem cell transplants is rejection by the recipient’s immune system. To evade tissue rejection, patients undergo immunosuppressive treatment, that makes them susceptible to microbial infections²⁷.

Inducing pluripotent cells directly from the patient’s cells to generate graft or tissue may resolve the problem associated with immunological rejection to an extent. However, low frequency of induced pluripotent stem cells is a major hurdle.

Behavior

Embryonic stem cells divide indefinitely and could induce tumor growth. Somatic cells can directly reprogram to specialized cells without the intermediate pluripotent state. While induced pluripotency of somatic cells can bypass the pluripotent state,  limited proliferative and lineage potential of resulting cells limit the scope²⁸.

Safety

Stem cells being used in cell therapy or regenerative medicine could be exposed to microbes, which eventually could cause infectious diseases²⁸. The necessary preliminary diagnostic test must be developed before the treatment. In addition, retaining intended biological activity before treatment is crucial for the success of the therapy. Systematic protocols need to be developed for isolation, testing and transplantation of stem cells, to ensure patients safety.

Conclusion

The awareness and interest of stem cell biology are expanding significantly. Over the past decade, several studies focused on understanding the complex nature of stem cell types. Investigations improving stem cell efficacy and stem cell migration²⁹ are underway. Apoptosis of engineered stem cells, once they have performed their role, is an active area of study³⁰. While research on applications of stem cells in tissue regeneration, genetic diseases, and cancer is growing there is still a long way before we witness widespread use of stem cells in therapy. Newly discovered gene editing technologies like CRISPR could advances stem cell research and offer enormous promise in treating multiple disorders.

References

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