Stem cells

Mala Parab

Definition of stem cells: stem cells are unspecialized cells that have two Simposium Stem Cell di defining properties: FKUI, ULTAH Kalbe 40 the ability to differentiate into other cells and the ability to tahun, 2 Sept.2006 self-regenerate/self-renew

Classification on Basis on Differentiation property

Totipotent all cell types. E.g.: zygote

Pluripotent all three germ layers. E.g.:

human embryonic stem cells

Multipotent many cell types. E.g.:

hematopoietic stem cells Unipotent can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem

Classification based on source/ origin

Stem Cell Types Based on The Source of Stem Cells

Embryonic stem cells obtained from the undifferentiated inner mass cells of a blastocyst, (an embryo that is between 50

to 150 cells)

Umbilical cord blood derived from the blood of the placenta and umbilical cord after birth

Adult stem cells undifferentiated cells found among differentiated cells of a specific tissue and are mostly Multipotent cells Bone marrow Adipose tissue CNS stem cells

Properties of adult stem cells

A stem cell possesses two properties:

Self-renewal, which is the ability to go through numerous cycles of cell division while still maintaining its undifferentiated state.

multipotency or multidifferentiative potential, which is the ability to generate progeny of several distinct cell types, (for example glial cells and neurons) as opposed to unipotency, which is the term for cells that are restricted to producing a single-cell type. However, some researchers do not consider multipotency to be essential, and believe that unipotent selfrenewing stem cells can exist.

These properties can be illustrated with relative ease in vitro, using methods such as clonogenic assays, where the progeny of a single cell is

characterized..

Adult stem cells express transporters of the ATP-binding cassette family that actively pump a diversity of organic molecules out of the cell..

Adult stem cell research has been focused on uncovering the general molecular mechanisms that control their self-renewal and differentiation.

Notch :The Notch pathway has been known to developmental biologists for decades. Its role in control of stem cell proliferation has now been

demonstrated for several cell types including haematopoietic, neural, and

mammary, stem cells.

Wnt :These developmental pathways are also strongly implicated as stem

cell regulators.

How Does Cell Therapy Work?

Stem cells can be used to

generate healthy and functioning

specialized cells, which can then replace diseased or dysfunctional

cells.

It is similar to the process of organ transplantation only the treatment

consists of transplanting cells

instead of organs.

How Does Cell Therapy Work?

Bone marrow transplants are an example of cell therapy in which the stem cells in a donor's marrow are used to replace the blood cells of the victims of leukemia. Cell therapy is also being used in experiments to graft new skin cells to treat serious burn victims, and to grow new corneas for the sight-impaired. In all of these uses, the goal is for the healthy cells to become integrated into the body and begin to function like the patient's own cells.

What Diseases Can be Cured by Stem Cell Therapies

Any disease in which there is tissue degeneration can be a potential candidate for stem cell therapies

1. Parkinson's and Alzheimer's diseases 2. Spinal cord injury 3. Stroke 4. Burns 5. Heart disease 6. Diabetes 7. Muscular dystrophy 8. Osteoporosis injuries 9. Cirrhosis hepatic 10. Leukemia 11. Sickle cell anemia 12. Osteoarthritis 13. Rheumatoid arthritis 14. Cancer

Stem Cell Characteristics Make Them Good Candidates for Cell-based Therapies

Potential to be harvested from patients High capacity of cell proliferation in culture to obtain large number of cells from a limited source

Ease of manipulation to replace existing non functional genes via gene transfer methods

Ability to migrate to host's target tissues, e.g. the brain Ability to integrate into host tissue and interact with surrounding tissue

Embryonic Stem Cells for Therapy

Advantages: Easily available from fertility clinics Pluripotent have ability to differentiate into cells derived from all 3 germ layers but not the embryonic membranes Immortal proliferate in culture & maintained in culture for several hundred doublings Disadvantages: Tumorigenic any contaminating undifferentiated cells could give rise to cancer Always allogenic immune rejection Ethically controversial

Adult Stem Cells for Therapy

Advantages: Can be taken form patients own cells not rejected by the immune system Already somewhat specialized: Inducement may be simpler Less ethical problems Disadvantages: Rare in mature tissues difficult to obtain in large quantities They don't live as long in a culture as embryonic stem cells Generally limited to differentiating into different cell types of their tissue of origin even though plasticity may exist

Skin Replacement

The knowledge of stem cells has made it possible for scientists to grow skin from a

patient's plucked hair. Skin (keratinocyte) stem cells reside in the hair follicle and
can be removed when a hair is plucked.

These cells can be cultured to form an epidermal equivalent of the patients own

skin and provides tissue for an autologous graft, bypassing the problem of
rejection.

It is presently being studied in clinical trials as an alternative to surgical grafts used

for venous ulcers and burn victims

Brain Cell Transplantation

The identification and localization of neural stem cells, both embryonic and adult, has been a major focus of current research.

Potential targets of neural stem cell transplants include stroke, spinal cord injury, and neurodegenerative diseases such as Parkinson's Disease.

Stem cells can provide dopamine - a chemical lacking in victims of Parkinson's Disease

Over 250 patients have already been transplanted with human fetal tissue

Cell-based therapy for Parkinsons Disease

Dopamine-Neuron Transplantation

Treatment of Diabetes

Recently, insulin expressing cells from

mouse stem cells have been generated. In

addition, the cells self assemble to form structures, which closely resemble normal pancreatic islets and produce insulin

Future research will need to investigate how

to optimize conditions for insulin production with the aim of providing a stem cell-based therapy to treat diabetes to replace the constant need for insulin injections

Bone/Cartilage Regeneration

Osteocel 1st commercial product in US: bone matrix containing allogenic human mesenchymal stem cells Launched in 2005 by Osiris Therapeutics

Other brands: Chondrogen, Tissue Repair Cells (TRCs), Prochymal, and Mesoblast

Drug Screening
Two of the worlds most successful blockbuster biotechnology products Epogen &

Neupogen were discovered & developed through the use of in vitro assays
involving blood stem cell technologies
Stem cells which can be expanded ex vivo and differentiated into specialized cell

types could enable the development of high-throughput screens for testing the
effects and possible toxicity of a range of drugs early in the drug development pipeline

Stem cells Blindness

In clinical trials at Moorfields Eye Hospital in London, surgeons restored eye sight for six patients who lost their sight after chemical accidents and genetic diseases. The patients went under successful stem-cell transplant.

Limbal stem Cell therapy

o

The treatment is known as limbal stem cell therapy, and the patients who received the treatment suffered from chemical burn or genetic disease know as aniridia

By replacing the limbal stem cells, the cornea begins to clear up as the cells are replaced with the healthy transparent layer again.

Neural Stem Cells

Neural stem cells (NSCs) are self-renewing, multipotent cells that generate the main phenotypes of the nervous system. They are located in: Sub-ventricular zone lining the lateral ventricles, where they give rise to newly-born neurons that migrate to the olfactory bulb via the rostral migratory stream and Subgranular zone, part of the dentate gyrus of the hippocampus NSCs primarily differentiate into neurons, astrocytes, and oligodendrocyte. NSCs are stimulated to begin differentiation via exogenous cues from the microenvironment, or stem cell niche. This capability of the NSCs to replace lost or damaged neural cells is called neurogenesis. The ependymal cells and astrocytes form glial tubes are used by migrating neuroblasts. The astrocytes in the tubes provide support for the migrating cells as well as insulation from electrical and chemical signals released from surrounding cells. The astrocytes are the primary precursors for rapid cell amplification. Neurons usually show radial migration pattern.

Section of the hippocampus, blue dots are neural stem cells

Mature neuron (red)

Functions of NSCs during differentiation:

Radial, astrocytes-like, GFAP-positive cell model:

The niche composed of blood vessels, astrocytes, microglia, ependymal cells, and extracellular matrix promote the quiescent state (Type B) of NSCs.

Once activated, the Type B cells develop into Type C cells, active proliferating
intermediate cells, which then divide into neuroblasts consisting of Type A cells. The undifferentiated neuroblasts form chains that migrate and develop into mature neurons. In the olfactory bulb, they mature into GABAergic granule neurons, while in the hippocampus they mature into dentate granule cells.

NSCs play a vital role during development producing the enormous diversity of neurons, astrocytes and oligodendrocyte in the developing CNS.

Function of NSCs during diseases:

NSCs are involved in migration and replacement of dying

neurons

Hippocampal stem cells migration elicited by SDF-1a, a chemokine during stroke helps to repair the injury

responses during stroke, multiple sclerosis, and Parkinson's

disease in animal models and humans is part of the current

investigation

Used to treat neurodegenerative disorders and spinal cord injury.

Hematopoietic stem cells

Hematopoietic stem cells (HSCs) are the blood cells that give rise to all the other blood cells. They give rise to all the blood cell types: Myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/ platelets, dendritic cells) Lymphoid (T-cells, B-cells, NK-cells) HSCs contain cells with long-term and short-term regeneration capacities and committed multipotent, oligopotent, and unipotent progenitors. HSCs are found in the bone marrow of adults, with large quantities in the pelvis, femur, and sternum and can mobilize out of the bone marrow into circulating blood Markers: CD 34+, CD 59+, Thy+, CD 38, C-Kit

Sources of HSCs:

1.

Bone Marrow: About 1 in every 100,000 cells in the marrow is a

long-term, blood-forming stem cell. Clinically used for bone marrow transplants.

2.

Peripheral blood: It has been known for decades that a small number of stem and progenitor cells circulate in the bloodstream, but in the past 10 years, researchers have found that they can coax the cells to migrate from marrow to blood in greater numbers by

injecting the donor with a cytokine, such as granulocyte-colony

stimulating factor (GCSF). Clinically used for autologous and allogenic transplants blood cell transplants.
3.

Umbilical Cord Blood: There have been suggestions that umbilical cord blood contains stem cells that have the capability of developing cells of multiple germ layers (multipotent) or even all germ layers, e.g., endoderm, ectoderm, and mesoderm (Pluripotent). Have been

used in treatment of Fanconi anemia

4. Fetal Hematopoietic System: Hematopoietic cells appear early in the development of all vertebrates. the earliest hematopoietic activity is indicated by the appearance of blood islands in the yolk sac (day 7-8) and in the AGMthe region where the aorta, gonads, and fetal kidney (mesonephros) begin to develop. an HSC in the bone marrow has four actions in its repertoire: 1) it can renew itself, 2) it can differentiate, 3) it can mobilize out of the bone marrow into circulation (or the reverse), or 4) it can undergo programmed cell death, or apoptosis. Understanding the how, when, where, which, and why of this simple repertoire will allow researchers to manipulate and use HSCs for tissue and organ repair.

Clinical Uses of HSCs: 1. Leukemia and Lymphoma: Among the first clinical uses of HSCs were the treatment of cancers of the bloodleukemia and lymphoma, which result from the uncontrolled proliferation of white blood cells. In these applications, the patient's own cancerous hematopoietic cells were destroyed via radiation or chemotherapy, then replaced with a bone marrow transplant, or, as is done now, with a transplant of HSCs collected from the peripheral circulation of a matched donor. 2. Inherited Blood disorder: treatment of hereditary blood disorders, such as different types of inherited anemia (failure to produce blood cells), and inborn errors of metabolism (genetic disorders characterized by defects in key enzymes need to produce essential body components or degrade chemical byproducts). The blood disorders include aplastic anemia, beta-thalassemia, BlackfanDiamond syndrome, globoid cell leukodystrophy, sickle-cell anemia, severe combined immunodeficiency, X-linked lymphoproliferative syndrome, and Wiskott-Aldrich syndrome. Inborn errors of metabolism that are treated with bone marrow transplants include: Hunter's syndrome, Hurler's syndrome, Lesch Nyhan syndrome, and osteopetrosis.

3.Rescue for Cancer Chemotherapy: cancer patients can be given an autologous stem cell transplant to replace the cells destroyed by chemotherapy. Once the drugs have washed out of a patient's body, the patient receives a transfusion of his or her stored HSCs. Epithelial Stem cells:

These give rise to epithelial cells which constitute 60 percent of the differentiated cells in the body. Responsible for covering the internal (i.e. intestinal lining) and external surfaces (i.e. skin) of the body, including the lining of vessels, glands, and other cavities. Epithelial stem cells are also found in the bulge region of the hair follicle

In skin epithelial stem cells are usually located in the basal lamina, near the dermal region. Based on the proliferative and morphological characteristics, Potten (1974) coined the term epidermal proliferative unit (EPU) to describe skin stem cells. The EPU is a stratified squamous epithelium constituted by different layers of cells. The innermost layer, called the basal layer, is strongly attached to its underlying dermis and contains mitotically active progenitor cells that divide and give rise to the differentiated suprabasal cells, which eventually differentiate in to different component cells of skin. The IFE continuously self-renews throughout life to replace the cells that are constantly sloughed off from the skin surface. In mice and humans, it takes about 3 to 4 weeks to replace all the cells of the IFE.

Applications of Skin Stem cells: 1. Skin stem cells for treatment of burns: Human keratinocyte SC cultured on fibroblast feeder layers, have an enormous proliferation potential, and only few cells can regenerate sufficient keratinocytes to cover the all-human skin surface. These cultured human keratinocyte SC can differentiate and reform a functional skin barrier that can be transplanted into patients suffering from severe burnt injuries.

Cellular therapy for inherited genetic diseases: Skin is a highly accessible source of SC, which have an extraordinary capacity of cellular expansion in culture, and that can be stably genetically modified. here are many different genetic diseases affecting the skin epidermis. Among the most devastating genetic skin diseases are the epidermolysis bullosa, which is characterized by an extreme fragility of the skin that results in painful blistering lesionss and require long time to heal. ue to permanent state of wound healing that stimulates SC proliferation, these patients are at high risk of developing skin cancer.
2.

The group of De Luca, have demonstrated that correction of a severe form of epidermolysis bullosa could be achieved by transplantation of genetically modified keratinocyte SC.

They isolated keratinocyte SC from a patient presenting epidermolysis bullosa caused by mutation in the gene encoding laminin 5, a critical component of hemidesmosomes. They expanded these skin SC in vitro and transduced these cells with a retrovirus expressing Laminin 5. They isolated cells stably expressing Laminin 5 and made genetically corrected cultured epidermal grafts that have been transplanted onto the patient skin lesions.. The genetically corrected skin tissue healed perfectly well and biopsies from the graft demonstrate that the recombinant skin presents a normal histology and the expression of the transgene remains stable even a year after the treatment.

Intestine Stem cells: The gastrointestinal epithelial stem cell niche is believed to be formed and maintained by the underlying cells of the mesenchymal lamina propria and their secreted basement membrane factors, which regulate stem cell behaviour through the paracrine secretion of various growth factors and cytokines, the receptors for which are situated on gastrointestinal epithelial cells The epithelial stem cell lineages of gastrointestinal tract undergo constant turnover, with complete self renewal every 2-7 days under normal circumstances and increases following tissue damage. The Notch signaling pathway and E2F transcription factor regulates gastrointestinal epithelial cell fate and differentiation of the four specialized epithelial lineages of the gastrointestinal tract. Increased levels of Notch protein negatively regulate the transcription of the Math1 gene, a basic loop-helix transcription factor, via up regulation of the Hes1 transcriptional repressor.

Molecular markers for identification of GISCs: LGR5, BMI1,CD133 are markers of crypt stem cell population. The GISCs are proposed to be used for treatment of colon cancer, chronic ulcers and transplants for small intestine. Mammary stem cells: The mammary gland in humans and in other mammals is a dynamic organ that undergoes significant developmental changes during pregnancy, lactation, and involution. Under the regulation of systemic hormones, as well as local stromal epithelial interactions, these cells proliferate extensively, differentiate during each pregnancy and lactation, and undergo apoptosis during mammary involution the mammary gland is subjected to major changes in morphology during distinct developmental windows

In humans, the mammary epithelium consists of a network of ducts that form before birth, by branching and invading the mammary fat pad. The ducts are formed by a basal layer of contractile, myoepithelial cells and a luminal layer of specialized epithelial cells. During puberty, ductal outgrowth rapidly increases under hormonal stimulation, resulting in side branching. The nal differentiation stage is achieved in the mammary gland during pregnancy and lactation, when numerous lobulo-acinar structures containing the milk-secreting alveolar cells are formed through extensive proliferation, followed by terminal differentiation. Cessation of lactation following weaning is accompanied by massive apoptosisand tissue remodelling, and the gland reverts to a structure resembling that before pregnancy.

The existence of self-renewing, multipotent mammary stem cells was rst suggested decades ago by the work of Daniel et al. Their studies in mice and rats demonstrated that an entire mammary gland can be generated from serially transplanted random fragments of epithelium. Mammary stem cells are usually represented as a population of small light cells without polarity. Cellular Markers: ESA+ , MuC1-, , alpha 6 integrin+, CD10+,

Table: 3. Fibroblast growth factor receptor 1 I]Examples of genes with overlapping expression 4. Tenascin C patterns in mammospheres, III]Genes upregulated in neurospheres, mammospheres and haematopoietic stem cells haematopoietic stem cells and embryonic stem cells 1. Notch 3 1. Integrin, beta 1 2. Latent transforming 2. Growth hormone receptor growth factor beta binding 3. Platelet-derived growth protein 3 factor receptor, beta 3. Apolipoprotein E polypeptide 4. GATA-binding protein 2 4. p53 target zinc nger 1V] Genes upregulated in protein mammospheres and 5. Policystic kidney disease embryonic stem cells 2 1. Nidogen 1 and 2 II.] Genes upregulated in mammospheres and 2. Glypican 4 3. Insulin-like growth factor neurospheres binding protein 4 and 7 1. p59fyn (FYN) oncogene 2. ATP-binding cassette, 4. WNT1 inducible signalling pathway protein 1 subfamily A, member 1

The Notch trans-membrane receptor proteins are part of a signaling pathway that is critical for the correct developmental fate of cells and various tissues. Activation of the Notch pathway results in changes of cell fate, such as proliferation of undifferentiated cells, blockage of differentiation or differentiation along a particular lineage. The vertebrate Notch 4 gene has been shown to be involved in normal mammary development. In vitro, over-expression of the constitutively active form of Notch 4 inhibits differentiation of normal breast epithelial cells. In vivo, transgenic mice expressing a constitutively active form of Notch 4 in the mammary gland fail to develop secretory lobules during gestation, and subsequently develop mammary tumours. Thus alterations in Notch 4 signalling might play a significant role in breast-cancer development

1.

2. 3.

4.

Apart from Notch 4, over expression of Wnt signaling pathway, - catenin, factors like APC, TCF-1 in mouse mammary glands increased MMTV promoter increases mammary tumor formation. LIF and PTEN are two other proteins involved in tumorogenesis of mammary glands. Applications: In-vitro developed mammospheres have been used as models to study evolution of breast cancers. Such models enables study of chemotherapeutic agents on receptors like estrogens, BRCA-1 and integrin mediated signal transductions. It allows one to bring about genetic manipulations followed by transplants as a part of treatment . Recent research demonstrated that mammary stem cells respond to both hormone withdrawal and stimulation. Thus antihormone therapy, can be used as a treatment aimed for preventing breast cancer recurrence Mammary stem cells have been shown to repopulate the fat pad of mammary glands post intensive cancer therapy.

In the presence of female hormones, the mammary stem cell outgrows to generate an extensive mammary tree (left). Following hormone deprivation, the mammary stem cell has reduced outgrowth potential (right).

In the presence of female hormones, the mammary stem cell outgrows to generate an extensive mammary tree (left). Following hormone deprivation, the mammary stem cell has reduced outgrowth potential (right). Prostate Stem cells: Prostatic epithelium is composed of multiple differentiated cell types, including basal, luminal (secretory), and neuroendocrine cells. Luminal secretory cells make up the majority of the epithelial layer and because they express androgen receptors (ARs), they can respond directly to androgens by simulating production and secretion of prostatic proteins, such as prostate-specific antigen (PSA) and prostatic acid phosphatase. In the human prostate, basal cells form a continuous layer, whereas in other species they are more scattered in appearance. Prostatic epithelial cells are identified by their morphological appearance, location, and also distinct patterns of marker expression. Basal cells express cytokeratins (CKs) 5 and CK14

Linear, bi-directional and lineage differentiation models are three models proposed for in-vivo development of prostrate glands. A diagnostic feature of human prostate cancer is the loss of basal cells. Prostate cancer can potentially arise from oncogenic transformation of CK5C8K basal cells resulting in rapid differentiation to a luminal phenotype, or alternatively from stem or multipotent progenitor cells within the CK5C8C intermediate or CK5K8C luminal populations where stem cells or CARNs are proposed to reside. Multiple lines of evidence demonstrate initiation of prostate cancer from luminal (and possibly intermediate) cells, based on targeted gene disruption by Cre-recombinase under the control of the probasin (either probasin or ARR2/probasin) or PSA promoters that show luminal-specic expression.

A body of work by the Witte Laboratory provides evidence to suggest that basal cells can initiate preneoplastic and cancerous lesions. For example, loss of PTEN negatively regulates p63C prostatic basal cell proliferation without blocking differentiation, resulting in an expansion of a prostate stem/progenitor-like subpopulation, using defined cell lineage markers including Sca-1C, Bcl-2C, and CD49fhi cells.

Bone and Muscle Stem cells

Skeletal muscle is a dynamic tissue that is capable of responding to physiological stimuli (i.e., intense exercise training) or a severe injury by mounting a well orchestrated regenerative response that restores the cytoarchitecture within a 2-wk period. The capacity for this regenerative response is primarily due to a mononuclear cell population termed satellite cells, usually present in peripheral (or satellite) position in relation to the adjacent, larger multinucleated myofiber. In response to injury or disruption of the basal lamina, the satellite cells become activated and have a remarkable proliferative capacity. Ultimately, the satellite cells either fuse to form multinucleated myotubes or re-establish a residual pool of quiescent satellite cells that have the capability of supporting additional rounds of regeneration

Skeletal Muscle development during embryogenesis Skeletal muscle formation is derived from the paraxial mesoderm early during vertebrate embryogenesis. The paraxial mesoderm coalesces to form segmented, epithelial spheres referred to as somites that occupy paired structures on either side of the neural tube. The somite is further specialized to produce the dermomyotome the sclerotome. These progenitors migrate beneath the dermomyotome to form the myotome and adopt a skeletal muscle fate. In addition, proliferating cells (Pax3+Pax7+ cells) from the central dermomyotome migrate directly to the myotome and continue to proliferate without expression of differentiation markers such as members of the MyoD family. MyoD transcriptional factors are expressed in somites and established skeletal muscle during embyogenesis

In adults muscle stem/precursor cells including the myogenic satellite cell population originate principally quail cells, bone marrow cells, somite and vascular components like endothelium, pericyte, mesoangioblast Satellite cells lack expression of the myogenic regulatory factors, including members of the Myod family A tyrosine kinase receptor (Met, for hepatocyte growth factor) is expressed broadly during development in the myotome of the somite and neural crest derivatives and is localized to the quiescent satellite cell population in adult skeletal muscle These studies demonstrate that myogenic stem/progenitors are capable of generating nonmuscle lineages including blood derivatives, vascular components, osteoblasts, and adipocytes in response to specific signals

For example, satellite cells that are cultured in the presence of bone morphogenic proteins (i.e., Bmp2) promote the osteogenic molecular program with an induction of the master transcriptional regulators Runx2 and Osterix Morphological analysis reveals interstitial edema with neutrophils that release trophic factors to activate the satellite cells within 2 h of injury Following activation of the satellite cell population, they re-enter the cell cycle and demonstrate a significant proliferative capacity between 2 and 3 d following injury This proliferative period is followed by a differentiation phase where myoblasts withdraw from the cell cycle and form small basophilic centronucleated myotubes (the hallmark of skeletal muscle regeneration) Fusion of myoblasts and further growth of the newly regenerated centronucleated myofibers ultimately results in restoration of the cellular architecture within an 2-wk period

Role of Stem cells in Disease and aging Congenital and acquired myopathies are common and deadly. The most common lethal myopathic disease is DMD, which is an Xlinked recessive disease. Studies indicate that mutations within Lamin A are associated with Emery- Dreifuss and Limb-Girdle muscular dystrophy patients. Age-related myopathies (i.e., sarcopenia) also have impaired regenerative defects The impaired proliferative capacity of satellite cells that reside in aged muscle has been shown to be due, in part, to perturbed Notch signaling, as forced expression of activated Notch restored the regeneration capacity of old (or aged) muscle The study of these cells shall thereby aid an sound understanding of somatic stem cell biology and serve as a platform for cell-based therapies directed toward patients with congenital birth defects and debilitating myopathies