Adult Stem Cells

(1)

Adult Stem Cells

Week 6

(2)

(3)

History of Adult Stem Cell Research

• 1940; first adult stem cell experiments

• 1950; reformation of blood cells in mouse by the infusion of bone marrow cells

• 1960; two different kinds of stem cell population have been identified in bone marrow; Heamotopoetic stem cell and bone marrow stromal cells)

• 1960; Newly formed neural cells from the brain

• 1990; formation of astrocyte, oligodendrocyte and neuron from the neural stem cells

(4)

Adult stem cell

• An adult stem cell is an undifferentiated cell that occurs in a differentiated (specialized) tissue, renews itself, and becomes

specialized to yield all of the specialized cell types of the tissue from which it originated.

• Adult stem cells usually divide to generate progenitor or

precursor cells, which then differentiate or develop into

"mature" cell types that have

characteristic shapes and

specialized functions.

(5)

• Adult stem cells are rare, difficult to identify and purify, and, when grown in culture, are difficult to maintain in the undifferentiated Their primary functions are to maintain homeostasis and, with limitations, to replace cells that die because of injury or disease

• Furthermore, adult stem cells are dispersed in tissues throughout the mature animal and behave very

differently, depending on their local environment.

• Current methods for characterizing adult stem cells are dependent on determining cell surface markers and

observations about their differentiation patterns in test

tubes and culture dishes

(6)

Sources of adult stem cells

• Bone marrow,

• blood,

• eye,

• brain,

• skeletal muscle,

• dental pulp,

• liver,

• skin,

• the lining of the gastrointestinal tract,

• pancreas.

(7)

Plasticity of ASCs

• Studies suggest that at least some adult stem cells are multipotent.

– stem cells from the bone marrow, a mesodermal tissue, can give rise to the three major types of brain cells, which are ectodermal derivatives

– stem cells from the brain can differentiate into blood cells and muscle tissue

• It is not clear whether investigators are seeing adult stem cells that truly have plasticity or whether some tissues contain several types of stem cells that each give rise to only a few derivative types.

(8)

Hematopoietic Stem Cells (HSCs)

(9)

• The hope that many diseases can someday be treated with stem cell therapy is inspired by the historical success of bone marrow transplants in increasing the survival of patients with leukemia and other cancers, inherited blood disorders,

and diseases of the immune system

• Moer than 40 years ago, the cell type

responsible for those successes was identified as the hematopoietic stem cell (Till and

McCullough, 1961)

(10)

• The ability of hematopoietic stem cells (HSCs) to self-renew continuously in the marrow and to differentiate into the full complement of cell types found in blood qualifies them as the premier adult stem cells

• With more than 50 years of experience, no other type of stem cell, adult, fetal or

embryonic, has attained such status.

(11)

Blood Cell Differentiation from Hematopoietic Stem Cells

• HSCs normally divide to generate either more HSCs

(self-renewal) or progenitor cells, which are precursors to various blood cell types.

• HSCs are found mainly in bone marrow, although T cells

develop in thymus, and some other cell types develop from blood monocytes.

• Once HSCs partly differentiate into progenitor cells, further differentiation into one or a few types of blood cell is irreversible.

(12)

Plasticity of HSCs

• There is a growing body of evidence that HSCs are plastic.

– HSCs can give rise to liver cells

– cardiac and muscle tissue formation after bone marrow transplantation in mice

– the development of neuron-like cells from bone marrow

– a single HSC transplanted into an irradiated mouse generated not only blood components (from the

mesoderm layer of the embryo), but also epithelial

cells in the lungs, gut, (endoderm layer) and skin

(ectoderm layer)

(13)

Major barriers to progress in HSC research

• Obtaining purified HSCs is a major challenge, and purification in a clinical setting is expensive and difficult. Because HSCs look and behave in culture like ordinary white blood cells, this has been a difficult challenge and this makes them difficult to identify by morphology (size and

shape).

• It has not been possible to culture HSCs in vitro,

although recent studies of mouse HSCs grown

in combination with components of the bone

marrow have offered some preliminary promise

(14)

Gold standard for HSCs

• The cells are injected into a mouse that

has received a dose of irradiation sufficient to kill its own blood-producing cells.

• If the mouse recovers and all types of blood cells reappear (bearing a genetic marker from the donor animal), the

transplanted cells are deemed to have

included stem cells.

(15)

Two kinds of HSCs

• If bone marrow cells from the transplanted

mouse can, in turn, be transplanted to another lethally irradiated mouse and restore its

hematopoietic system over some months, they are considered to be long-term stem cells that are capable of self-renewal.

• Other cells from bone marrow can immediately

regenerate all the different types of blood cells,

but under normal circumstances cannot renew

themselves over the long term, and these are

referred to as short-term progenitor or precursor

cells.

(16)

(17)

Sources of Hematopoietic Stem Cells

• Bone Marrow

Gray's Anatomy of the Human Body

(18)

Sources of Hematopoietic Stem Cells

• Peripheral Blood: 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).

• Umbilical Cord Blood

• Fetal liver

(19)

Stem Cell Niches

(20)

Hematopoietic stem cells: their niches

• The balance between self-renewal and

commitment of stem cells is controlled by a combination of cell-intrinsic and external

regulatory mechanisms.

• The intrinsic cellular and molecular properties of stem cells have been characterized extensively, and recently, the ‘niches’ or specific

microenvironments in which stem cells reside in situ have been studied at the molecular level.

• The concept of the stem cell niche was first

proposed for the human hematopoietic system in

the 1970s.

(21)

Structure of HSC niche

• The hematopoietic niche is conceptually divided into three parts:

– an osteoblastic zone (located near osteoblasts),

– a vascular zone (near the sinusoids)

– cells of neighboring HSCs

(22)

The niche

that regulates the birth and differentiation of blood-forming HSCs

• Depleting osteoblasts of a receptor for bone

morphogenetic protein (BMP) caused a doubling in both the osteoblast population and the stem-cell population.

• expansion of the stem-cell population when they

increased the numbers of osteoblasts by using

parathyroid hormone (PTH).

Nature 425, 836–841 (2003)

(23)

HSC niche.

• The HSC niche is located primarily on the surface of trabecular bone, where a small subset of spindle- shaped N-cadherin-positive

osteoblastic cells (indicated as

SNO cells) are the key component of the HSC niche.

• N-cadherin and β-catenin form an adherens complex at the interface between stem cells and niche

cells, assisting stem cells in attaching to the niche.

• Multiple growth factors and cytokines are involved in stem- niche interaction. (SCF/Kit, Jagged/Notch, SDF-1/CXCR4, and Ang1/Tie2)

Annu. Rev. Cell Dev. Biol. 2005

(24)

BM cells consist of two HSCs

• Resting HSCs:

– Resting HSCs are maintained in a quiescent state as a result of their close proximity to osteoblasts.

• Actively dividing HSCs.

(25)

The HSCs in the niche

• Quiescent HSCs (SP: side

population) adhere to the niche through cell-adhesion

molecules, such as N-

cadherin. Spindle-shaped osteoblastic (SNO) cells constitute the HSC niche.

Activated HSCs (non-SP)

generate HSCs (self-renewal) and progenitor cells

(differentiation) by asymmetric cell division. HSCs lose the abilities of multi-potency and self-renewal, following their cell division and differentiation.

Suda 2005

(26)

The HSCs in the niche

• Quiescent HSCs (Tie2+N-

cadherin+c-Kit+Sca-1+lineagẽ

SP cells) are regulated by the signaling of receptors,

cytokines and cell adhesion molecules (e.g. integrins and cadherins), which are produced by SNO cells. Ang-1 produced by SNO cells activates Tie2 on HSCs and promotes tight

adhesion of HSCs to the niche, resulting in quiescence and enhanced survival of HSCs.

Tie2–angiopoietin signaling also upregulates N-cadherin expression by HSCs.

Suda 2005

(27)

Side population cells

• in 1996, Goodell et al reported on a new method for the isolation of hematopoietic stem cells (HSCs) based on the ability of the HSCs to efflux a fluorescent dye.

• Cells subjected to Hoechst 33342 dye staining and fluorescence activated cell sorting (FACS) analysis can give a fluorescent profile

• Those that actively efflux the Hoechst dye appear as a distinct population of cells on the side of the profile; hence the name the

‘side population’ (SP) has been given to these cells. Typically, SP cells comprise about 0.05% of the total bone marrow cells and are highly enriched for long-term repopulating activity

Goodel 1996

(28)

Changes in SP cells and non-SP cells in KSL (Kit·Sca-1·Lin·) cells after 5-FU treatment

• In control KSL cells, 19.4±0.7% of cells are in SP,

whereas 2 days after 5-FU treatment, most cells in the KSL fraction are SP (70.1±6.2%) and the number of MP cells is reduced dramatically

Suda 2005

(29)

Assays for hematopoietic stem cells (HSCs)

• The CFU-S assay relies on the fact that, post transplant into an irradiated

recipient mouse, stem cells in the transplanted marrow seed onto the surface of the spleen and form macroscopic colonies of mature haemopoietic cells. By counting the

number of stem-cell-derived colonies on the spleen surface, an estimate of the number of stem cells in the transplanted population can be obtained.

• Additionally, the CFU-S assay can be scored at various times to detect stem cells with varying degrees of

primitiveness. Thus,CFU-S scored eight days post transplant (CFU-S d8) are more mature than CFU-S d12, which in turn are more mature than the pre-CFU- S cells.

Whetton 1999

(30)

Assays for HSCs

• the long-term marrow culture- initiating cell (LTCIC) and

cobblestone area forming cell (CAFCs) assays are in vitro assays that detect very

primitive components of the stem cell compartment.

• Studies on the different cells within the stem cell

compartment indicate that the more primitive cells have

greater potential for sel renewal than those further along the ‘age’ hierarchy.

Denning-Kendall 2003

(31)

Human HSC detection

• Definition of a long-term or short-term repopulating cell is clearly impossible in the human system; however,

human stem cells have been defined on the basis of

their activity in a range of in vitro assays similar to those described for murine stem cells.

• Additionally, a number of xenograft models for human

HSC detection, including the use of fetal sheep as a

xenograft recipient. However, by far the most common

xenograft model for the human HSC involves the use of

severe combined immunodeficient (SCID)/non-obese

diabetic (NOD) mice, an assay system that has enabled

an assessment to be made of the reconstitution abilities

of human progenitor cells.

(32)

The location of HSCs during development and in adulthood.

• Primitive haemopoietic stem cells can be found in the early yolk sac as the first evidence of haemopoiesis in the developing embryo.

• an intermediate site of stem cell activity exists in the embryo defined by its proximity to the developing aorta, gonad and mesonephros.

• From the AGM region, the stem cells migrate to the fetal liver, which is maintained as the principal site of haemopoietic activity throughout embryonic life.

• At or near birth, hematopoiesis moves from the fetal liver to the bone marrow, which is maintained as the primary site of hematopoietic activity in the adult animal.

Whetton 1999

(33)