Tissue Culture and Differentiation

Monolayer cells: Most cells tend to grow as monolayers (eg fibroblasts) on a substratum (such as a petri dish or flask). They grow up until reach 100% confluence.

Suspension: They are floating cells in the medium (eg haemopoeitic cells, hybridomas), cells in suspension continue to grow until all of the nutrients in the medium are exhausted or depleted.

A practical note: A pH indicator indicates that a change in medium is required (due to nutrient depletion or waste build up) and the cells should be passaged or subcultured.

Cell migration is one of the important phenomenon in monolayer cell cultures.

The Cell CycleThe Cell Cycle

• Four successive phases of standard eukaryotic cell cycle

• Cell is either dividing or in interphase

• 12 hours or longer

Different Types of Stem Cells

• Totipotent

Pluripotent

Multipotent

Destiny of cells:

(cell growth, immoralization, differentiation, apoptosis, cell death).

Cell growth: Cells in culture usually have a finite lifespan. Most divide about 50 times before they finally stop dividing.

Practical notes: It is advisable to freeze down early passage stocks of cells (to be thawed later) to avoid this limit.

Transformed cells can become tumorigenic if used in vivo and display a variety of altered and unique properties that may be unsuitable.

Transformation of cells with virus or oncogene can immortalise cells so that they continually divide (eg HeLa cells, hybridomas).

Cells may also differentiate in medium. Either spontaneously (eg PSMB embryonic stem cells) or under the influence of specific differentiation conditions (eg retinoic acid induces F9 embryonic stem cell differentiation). Again, growth characteristics may change.

Cell death: (apoptosis or necrosis)

Cell cultures: Primary and transformed

Primary cultures and finite cell lines usually are anchorage-dependent and contact inhibited; transformed cells typically are not.

Transformed cells

Primary or finite cells

Cell DifferentiationCell Differentiation

• All cells carry out basic functions – Respiration, growth, division, synthesis

• Most cells have specialized capabilities– 200 different cells in the human body.

• Cells in their final, differentiated statefinal, differentiated state usually have very unique characteristics, reflected in their morphology

Embryonic stem Cell differentiation:

1- Early differentiation

A- Cell differentiation (specialization) begins early in embryonic development – corresponds with loss of embryo’s dependence on maternal mRNA (i.e., coincident with turning on of the embryonic genome; usually about 4-cell+ stage) – individual embryonic cells (blastomeres) are no longer totipotent

B- Initial recognizable differentiation event involves formation of the blastocyst A: Inner cell mass (ICM or embryo proper, sometimes called embryonic disc) B: Trophectoderm (also called trophoblast)

C- Subsequent differentiation/specialization involves formation of the inner germ layer (endoderm) followed by formation of the middle germ layer (mesoderm)

D- All of the body tissues are developed from these 3 germ layers

E- Stem cells undergo self-renewing divisions (adult stem cells). Tissue-specific stem cells are found in adult tissues including blood, skin, central nervous system, liver, gastrointestinal tract, fat and skeletal muscle. They are responsible for regenerating damaged tissue and maintaining tissue homeostasis.

ES cells ---- Transient cells ---- Matured cells

Potentially Renewing Potentially Renewing CellsCells

• Not constantly renewing

• Return to active cyclingactive cycling (cell cycle/division) in response to critical cell depletion

• Renewal by simple duplicationsimple duplication of existing differentiated cells – division gives rise to daughter cells of

the same type

TRENDS in Biotechnology Vol.21 No.8 August 2003

2- Cell maturation/tissue specialization

A- Specificity and acquisition of tissue-specific functions

B- Hallmarks of cell differentiation:Loss of totipotency Loss of ability to divide

C- Cell differentiation and cell proliferation (growth) are intimately entwined

Transdifferentiation can be defined as a direct switch of an already differentiated cell to another type of differentiated cell.

3- Non-dividing cells

A- Q cells

Cells in G0 – thus reversibly withdrawn from the cell cycle

Can be induced to re-enter the cell cycle by growth factors, e.g.: (a) Tissue repair (wound healing) (b) Tissue renewal (e.g. partial hepatectomy), etc.

B- Terminally differentiated cells

Fully differentiated, but can no longer divide

Eventually die by a process termed apoptosis

For example, squamous of skin, goblet and brush-border cells of intestinal epithelium

Tumor Cells Can Be Induced to Differentiate, but the Differentiated Phenotype Is Reversible upon the Removal of the Inducing Agent Due to Rapid Hydrolysis of Inducing Signals or to Unresponsiveness of Regulatory Genes.

Tumor Cells Can Be Induced to Differentiate Terminally by Appropriate Agents if the Differentiation Gene Is Not Mutated; Conversely, Tumor

Cells with the Mutated Differentiation Gene Can Be Resistant to Terminal Differentiation.

Genes and differentiation:

PRASAD K.N. et al. 2001

TRENDS in Biotechnology Vol.21 No.8 August 2003