The Stem Cell Concept

Gastrulation(Establishment of the three germ layers)

Organogenesis(The process of organ formation)

During organogenesis the interactions between different parts of the embryo create privileged sites

“ stem cell niches” The stem cell niches provide extracellular matrix derived, juxtacrine and paracrine factors which allow the cells residing in them to remain relatively undeifferentiated such that they become the stem cells.

What is a stem cell?A relatively undifferentiated cell which when it divides produces:

1. At least one of two daughter cells that retains its undifferentiated character (self-renewal)

2. A daughter cell that can undergo further differentiation.

Two modes by which stem cells undergo self-renewal

and also produce differentiated cells

In many organs such as the adult bone marrow the stem cell lineages go through progressive restrictions in potency

Proof of concept: The Hematopoetic Stem Cell Till the mid-twentieth century it was thought that cell specification and differentiation were restricted to the early embryo and in the later stages only growth of existing parts occurred.

1960s: Investigation into how new blood cells (erythrocytes, granulocytes, platelets and lymphocytes) are constantly being produced to replace the ones destroyed by the spleen every day.

1961: Earnest McCulloch and James Till performed some elegant experiments to show that a common generative cell “the hematopoetic stem cell” gave rise to all the different types of blood cells.

The concept that the different blood cell types were continuously generated by hematopoietic stem cell was first proposed in 1909 by the Russian histologist Alexander Maximov.

He worked in St. Petersburg but his studies were curtailed by the Russian Revolution.

He is credited with coining the word “Stamzelle” (stem-cell) to refer to the regenerative capacities of the these cells.

Earnest McCulloch and James Till in 1961 injected bone marrow cells from a donor mouse into lethally irradiated mice (irradiation with X-ray was used to destroy their blood cell precursors) of the same genetic background. Some of the individual donor cells produced discrete nodules on the spleens of the host animals. These nodules contained erythrocyte, white blood cells and platelet precursor cells.

RADIATION RESEARCH 14, 213-222 (1961)

A Direct Measurement of the Radiation Sensitivity of Normal Mouse Bone Marrow Cells

J. E. TILL AND E. A. McCULLOCHDepartment of Medical Biophysics, University of Toronto, and the Divisions of Biological Research and Physics of the Ontario Cancer Institute,Toronto, Ontario

Experiments were performed in which the donor cells were irradiated to genetically mark each cell with random chromosome breaks to confirm that each of these different cell types in a nodule arose from a single cell.

Nature, Vol. 197, No. 4866, pp. 452-454, February 2, 1963

CYTOLOGICAL DEMONSTRATION OF THE CLONAL NATURE OF SPLEEN COLONIES DERIVED FROM TRANS-PLANTED MOUSE MARROW CELLS

By DR. A. J. BECKER, E. A. McCULLOCH and J. E. TILLDepartrnent of Medical Biophysics, University of Toronto and Ontario Cancer Institute, Toronto

For the “colony-forming cell” to be a true stem cell, it had to produce not only the differentiated blood cells but also more colony-forming cells. This was demonstrated by taking the nodule derived from a single genetically marked colony-forming cell and injecting the cells from this nodule into another irradiated mouse. Many spleen colonies emerged each of them having the same chromosomal arrangement as the original colony.

Two types of stem cellsEmbryonic stem cells Adult stem cells

Derived from the inner-cell mass of mammalian blastocysts

OR

From fetal gamete progenitor cells.

These are capable of producing all the cell types of the embryo.

Found in tissues and organs after the organ has matured.

These stem cells usually involved in repairing and regenerating tissues of that particular organ and can form only a subset of cell types.

Stem cell potency The ability of a particular stem cell to generate various types

of differentiated cells.

Totipotent cells are capable of forming every type of cell in the embryo as well as the trophoblast cells of the placenta

Pluripotent stem cells have the ability to become all the cell types of the embryo but not the trophoblast.

Multipotent stem cells are present in the embryo or the adult but they can generate a small subset of all possible cells of the body e.g. the hematopoietic stem cell

Unipotent stem cells are found in a particular tissue and can regenerate a particular type of cell e.g. spermatogonia can only form sperm.

Progenitor cells are different from stem cells because they are not capable of unlimited self-renewal. They have the capacity to only divide a few times before differentiating. A Progenitor cell is often produced when a stem cell divides.

Adult Stem CellsSeveral adult organs contain stem cells that can give rise to a limited set of cell and tissue types

Such cells are very difficult to isolate as they are present in very low numbers i.e.< 1 per 1000 cells in an organ. They also have a very low rate of proliferation. Nonetheless they have been used for therapeutic purposes.

Stem Cell NichesThe ability of a cell to become a stem cell is determined largely by where it resides

Continuously proliferating stem cells are housed in specialized regulatory microenvironments known as stem cell niches

Stem cell niches are highly complex and dynamic, including both cellular and acellular componentsAmy J. Wagers

The Stem Cell Niche in Regenerative Medicine

Cell Stem Cell, Volume 10, Issue 4, 2012, 362 - 369 http://dx.doi.org/10.1016/j.stem.2012.02.018

Stem cell microenvironments regulate proliferation vs differentiation via paracrine and juxtacrine factors produced by cells that make up the niche

For example in the C. elegans gonad the distal tip cell and its long projections provides the niche for the germline stem cells

The germ cell precursors near the distal tip cell divide mitotically forming the pool of germ cells but as they get farther away from the distal tip cell they enter meiosis.

Regulation of stem cell proliferation vs differentiation is very critical

Too much differentiation depletes the stem cell population and promotes the phenotype of aging and decay. Too

much proliferation can cause cancers to arise.

This balance is often maintained by antagonistic paracrine factors.

THE HEMATOPOIETIC STEM CELL NICHE The HSC niche is found in the hollow cavities of

trabecular bones where the bone marrow resides.

HSCs are very close to bone cells and endothelial cells that line blood vessels

A complex cocktail of paracrine factors Wnts Angiopoietin, Stem cell factor combine with cell surface signals from Notch and Integrins to regulate HSC proliferation and differentiation.

Hormones, blood pressure and neurotransmitters from adjacent axons also regulate hematopoiesis.

This way there is more production of WBCs when there is an infection and more production of RBC when one climbs to high altitudes.

Many types of stem cell niches harbour two kinds of stem cells i.e. rapidly cycling and quiescent

Wilson and colleagues have demonstrated that there are indeed two sub-populations of HSCs in the blood.

One subpopulation can divide rapidly in response to immediate needs, while a quiescent population is held in reserve and appears to consist of those cells that have the greatest potential of self-renewal.

Depending on conditions stem cells from one population can enter the other

Myeloproliferative disease is a cancer of the blood stem cells and their non-lymphocytic derivatives. It results from a failure of the niche to provide differentiation signals. These signals come from immature osteoblasts.

GERM CELL NICHES IN DROSOPHILA

The apical “hub” consists of 12 somatic cells to which are attached to about 5-9 germ-line stem cells The germ-line stem cell divides asymmetrically to form another germ-line stem cell (which remains attached to the

hub) and a gonialblast which will further divide to form sperm precursors (spermatogonia and spermatocyte cysts). Hub cells secrete the paracrine factor Unpaired. This activates the JAK-STAT pathway in the adjacent germ-line stem

cells to specify their self-renewal. Cells that are distant do not receive this signal and continue on the path of differentiation.

Physically the asymmetric division involves the tethering of one centrosome to the cortical cytoplasm at the point where the hub cell contacts the germline stem cell through specific cell adhesion molecules.

Stem cell niches in the testes of male Drosophila illustrate the importance of proximity and asymmetric cell division

THE INTESTINAL STEM CELL NICHE

The stem cells residing at the base of the intestinal crypts divide once a day. Some daughter cells become stem cells and stay in the crypt others proliferate as transient amplifying cells and

further differentiate into enterocytes, goblet cells and enteroendocrine cells. These cells migrate to the tip of the villus and undergo apoptosis.

THE INTESTINAL STEM CELL NICHE

The stem cell niche at the base of the crypt consists of Paneth cells (red) and stem cells expressing GFP under the control of Lgr5 gene promoter (green)

A confocal micrograph looking down into the bottom of the crypt shows paneth cells (red) in close association with stem cells (green)

Lineage tracing studies have shown that intestinal stem cells expressing the Lgr5 protein can generate all the differentiated cells of the intestine.

A single such stem cell grown in culture can produce a “mini gut” containing all the cell types.

A stem cell can generate its own niche by producing a special type of differentiated cell, the Paneth cells.

Paneth cells express several juxtacrine and paracrine factors such as Wnt3, Delta like 4, the ligand of Notch and epidermal growth factor etc.

Removing Paneth cells prevents the formation of all differentiated cell types.

The stem cells amplify by symmetric division and the daughters compete for interaction with Paneth cells. Only about half can bind to Paneth cells and these remain as stem cells

TOOTH REGENERATION AND LOST NICHES Some species of animals in the course of

evolution appear to have lost a stem cell niche while other related species retain them.

Rodent incisors are one such as they differ from incisors of humans and most other mammalian groups as they continue to grow throughout the life.

Each incisor has two stem cell niches one on the “inside” facing into the mouth and the other on the “outside” towards the lips. Most mammals lack these stem cells niches.

A balance of paracrine factors is at work regulating proliferation and differentiation. Stem cells are kept in the undifferentiated state by factors that include FGFs and BMP inhibitors. While BMPs and FGF inhibitors promote differentiation into enamel forming ameloblast cells.

The cervical loop of the mouse incisor is the stem cell niche for the enamel secreting ameloblast cells. These cells migrate from the base of the stellate reticulum into the enamel layer allowing the teeth to keep growing.

Mesenchymal Stem Cells: Multipotent Adult Stem Cells Most adult stem cells have limited ability to form only a few cell types. For example hematopoietic stem

cells labelled with GFP were placed in the mouse. Their labelled descendants were only found throughout the animals blood but not in other tissues.

Some adult stem cells have a surprisingly large degree of plasticity e.g. the Mesenchymal Stem Cells (MSCs) also known as Bone marrow-derived stem cells (BMDCs). However their potency remains a controversial issue.

Originally found in the bone marrow, MSC have now also been found in fat, muscle, thymus and dental pulp as well as in the umblical cord and placenta.

Some physicians advocate the freezing of umblical cord blood or teeth as source of stem cells for therapeutic purposes later on in life.

These cells from mice have failed the test of “pluripotency” e.g. the ability to generate tissues of all three germ layers when inserted into a blastocyst

Mesenchymal Stem Cells Mesenchymal stem cells can give rise to cells of bone, muscle, cartilage and fat lineages.

The Differentiation of MSCs along a particular lineage is influenced by exposure to paracrine factors as well as cell matrix molecules.

Matrix components like laminin has been implicated in keeping MSCs in an undifferentiated state.

Certain paracrine factors drive differentiation down different lineages e.g. PDGF is critical for cartilage and fat differentiation, TGF-β was important for chondrogenesis and FGF signaling was crucial for bone cell formation.

The elasticity of the matrices on which these cells are grown also influences them. Human MSCs grown on collagen-coated matrices of elasticity 0-1Kpa, express neuronal markers Human MSCs grown on matrices of elasticity 10 Kpa express MyoD (a muscle marker). Human MSCs grown on matrices of elasticity 100 Kpa express CBFα1 (a osteogenic marker)

PLURIPOTENT EMBRYONIC STEM CELLSThese cells can generate all the 220 cell types present in the adult mammalian body

In the laboratory pluripotent stem cells can be derived from two sources:

1. The first source is the inner cell mas of the early mammalian embryo that gives rise to the embryo body as opposed to the outer cells that generate the placenta. The inner cell mass gives rise to the embryonic stem cells (ESCs).

2. The second source is the primordial germ cells which have not differentiated into sperm or eggs. When isolated from the embryos these cells divide remain diploid and are called embryonic germ cells (EGC).

The decision to be an inner cell mass (ICM) or trophoblast cell is made first. Prior to blastocyst formation all cells express Cdx2 and Oct4. After the decision to make either trophoblast or ICM each group of cells express their own set of genes.

The pluripotency of embryonic stem cells is maintained by a core of three transcription factors: Oct4, Sox2 and Nanog.

These proteins bind to the enhancers of their own genes to maintain their expression while at the same time activating one anothers enhancers.Trophoblast cells express Cdx2 which down

regulates Oct4 and Nanog.

INDUCING ES CELL DIFFERENTIATION

Driving the ES cells to adopt either a neuronal or glial cell can be achieved by changing the media in which these cells grow.

ES cells retain their ability to differentiate into different cell types even when introduced into the adult body.

EGC were able to cure motor neuron injuries by differentiating into neuronal cells as well as by producing factors like BDNF which prevented death of existing neurons.

ES cells from monkey blastocysts have been used to cure a Parkinson’s like condition in adult monlkeys whose dopaminergic neurons had been destroyed.

The importance of pluripotent stem cells in medicine is potentially enormous:

• Used to produce new neurons for people with degenerative brain disorders e.g. Alzheimer’s or Parkinson’s disease or spinal cord injuries.

• Used to produce a new pancreas for people with people with diabetes.

• To produce new blood cells for people with anemias.

• To replace damaged heart tissue with new heart cells.

These therapies are already working in mice.

However, one difference between human and mouse experimentation is that mice can be inbred and made genetically identical; humans obviously cannot.

As human ES cells differentiate, they express significant amounts of Major histocompatibility proteins that can cause immune rejection.

Other problem is ethical concerns about using human embryos as source of ES cells

To get around the problem of immune rejection human ES cells could be modified or somatic cell nuclear transfer could be used.

Therapeutic cloning:In this technique, a nucleus from the patient is inserted into an enucleated oocyte. The resulting embryo is grown in vitro until it has developed an inner cell mass. Cells from the inner cell mass are then cultured to generate stem cells that are genetically identical to the patient.

Therapeutic cloning has been shown to work in mice to restore dopaminergic neurons damaged by Parkinson’s disease.

Ethical concern: Growing human embryos

INDUCED PLURIPOTENT STEM CELLS (iPSCs):A wave of the future?

In 2006 Kazutoshi Takahashi and Shinya Yamanaka of Kyoto University demonstrated by inserting activated copies of four genes that nearly any cell in the adult mouse can be made into an induced pluripotent stem cell (iPSC)

Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006) 

These genes were Sox2 and Oct4 (which activates Nanog and others required for pluripotency).

c-Myc required opening up the chromatin to provide access to Sox2 and Oct4.

Klf4 which prevent cell death.

Shinya Yamanaka was awarded the Nobel prize in Physiology and medicine in 2012 for this work

Protocol for curing a “human disease” in mouse using iPScs

Studies are ongoing to see if such therapy could cure other human diseases such as diabetes, macular degeneration, Parkinson’s disease, Alzheimer’s disease as well as liver and heart diseases.

iPSCs can be used to create in-vitro models of human diseases to study them when animal models are not available

For example mice do not get the same type of cystic fibrosis that humans get.First mouse iPSCs were used to generate lung epithelia.

Subsequently iPSCs derived from human patients with cystic fibrosis have been generated which can be used to screen drugs to treat cystic fibrosis

Natural stem cell therapy: Co-operation between fetus and mother

Pluripotent and multipotent stem cells have been found in unexpected places e.g. in the blood of pregnant mice and women.

Multipotent stem cells from the fetus were found to integrate into the mother’s heart which was damaged surgically.