Chapter 17 Differentiation “ The phenotype of cells cultured and propagated as cell line is often different from that of the predominating cell type in the originating tissue ” Proliferation  Cell Growth

Proliferation and Differentiation “ Cell proliferation is incompatible with the expression of differentiated properties ” Malignant tumor cells: sometimes break the restriction  Melanin continues to be synthesized while the cells are proliferating

Differentiation “ The process leading to the expression of phenotypic properties characteristics pf the functionally mature cell in vivo ” “ The combination of Constitutive and Adaptive properties found in the mature cell ” (1) (2) Constitutive: stably expressed w/o induction Adaptive: with positive or negative regulation of expression

Terminal Differentiation “ A cell has progressed down a particular lineage to a point, that the cell cannot progress anymore ” Examples: Neurons, Skeletal Muscle Cells, Keratinocyte Exclusion: Fibrocytes 1.produce extracellular matrix 2.express the hematopoietic cell surface markers (CD34+ & CD45+) and fibroblast marker (collagen) 3.migrate to wound sites (wound healing)

Two main pathways to Differentiation 1. In constantly renewing tissues: --e.g., epidermis, intestinal mucosa, blood --Pluripotent undifferentiated stem cells  Unipotent Progenitor cells  Terminal differentiated cells --this process gives rise to mature, differentiated cells that normal will not divide 2. In tissues that do not turn over rapidly: --e.g., fibrocytes, blood vessel endothelial cells, glial cells, hepatocytes, satellite cells -- Cells lose their differentiated properties and reenter the cell cycle -- When the tissue has regained the appropriate cell density by division, cell proliferation stops and differentiation is reinduced. --This type of renewal is rapid

Whether the cells that reenter the cell cycle to regenerate the tissue are phenotypically identical to the bulk of the differentiated cell population ??

Cells in cell culture can lose properties they originally had, such as protein expression, or change shape Dedifferentiation When Dedifferentiation occurs: 1. Adaptive Process: regaining the differentiated phenotype under the right inducers 2. Selective Process: because of cell ’ s greater proliferative potential

Cell Properties 1.Irreversible characteristic: e.g., mature keratinocyte -- cessation of DNA synthesis 2.Reversible characteristic: e.g., hepatocyte -- re-induction of albumin synthesis Progenitor cells Differentiated cells Stem cells irreversible Reversible Differentiation Process

Commitment & Cell Lineage 1.A hematopoietic stem cell, after commitment to lymphocytic differentiation, would not change lineage at a later stage 2.Stem cell  Progenitor cell  Differentiated cell 3.Some progenitor cells can revert to stem cells with multilineage potential  such irreversible commitment must occur much later than “ the point ” 4.Most cultures are derived from normal tissues (stem cells, late progenitor cells, differentiated cells) do not alter to a different lineage 5.Tumor culture: changes its commitment  K562 cell line: isolated from a myeloid leukemia & be capable of erythroid differentiation  C6 glioma of rat: expresses both astrocytic & oilgodendrocytic features 6.Cell lines are regarded as a mixed population of stem cells, progenitor cells, and differentiated cells

Re-Differentiation (I) 1.The majority of cell lines do not express fully differentiated properties 2.Developing Organ Culture System: Advantages: (1) retain three-dimensional, high-cell-density tissue architecture (2) prevent dissociation (3) prevent selective overgrowth of undifferentiated cells Disadvantages: (1) inability to propagate large numbers of cells (2) heterogeneity of the tissue sample 3. In pure populations of cells: --to reinduce the differentiated phenotype by recreating the correct environment --to define individual influences exerted on the induction and maintenance of differentiation

Glial cells: oligodendrocyte & astrocyte CNS Schwann cells: 1. using cholera toxin as a mitogen 2. optimal condition: glial growth factor + forskolin + 10% serum Schwann cells ??

Re-Differentiation (II) Environment (I) 1.Brain (O2A common progenitor cell of oligodendrocyte and progenitor cell of type 2 astrocyte) -- in the mixture of PDGF and bFGF: remains a proliferating progenitor cell -- in the absence of growth factor or serum: differentiate into an oilgodendrocyte --in the fetal bovine serum or a combination of ciliary neurotropic factor (CNTF) & bFGF  differentiate into a type 2 astrocyte 2.Cardiac muscle cell: -- in serum and bFGF: remains undifferentiated and proliferative properties -- in the absence of serum: differentiate

Re-Differentiation (II) Environment (II) 3. MDCK cell (cell line from dog kidney) / salivary gland epithelium / mammary epithelium: --in the mixture of epimorphin and HGF (hepatocyte growth factor)  inducing tubule formation (differentiation) 4. Prostatic epithelium / epidermal epithelium: -- in the presence of KGF (keratinocyte growth factor): differentiation 5. Primitive embryonal stem (ES) cell: --differentiate Spontaneously --in bFGF & SCF (stem cell factor) & LIF (lymphocyte inhibitory factor)  kept in the undifferentiated proliferative phase

Stem Cell Plasticity 1.Unipotent stem cell: e.g., stem cell in the basal layer of dermis -- will give rise to only one lineage -- differentiate into a keratinocyte 2. Bipotent stem cell: e.g., a lymphoid stem cell -- will give rise to two lineages -- differentiate into T- or B-lymphocyte 3.Multipotent stem cell: e.g., stem cell in the bone marrow -- will give rise to more than two lineages -- differentiate into granulocytes, monocytes, megakaryocytes, mast cells, erythrocytes 4.Totipotent stem cell: e.g., embryonal stem cell (ES cell) -- will give rise to every cell lineages

Stem Cell 1.Non-regenerative tissues do have stem cells -- Brain (Neurons) 2.Tissue localization does not necessarily mean lineage commitment and reduced potency -- Liver stem cells can generate neurons -- Bone marrow stem cells can generate cardiac muscle cell, hepatocytes, neurons -- Muscle stem cells can generate hematopoietic cells -- Neural stem cells can generate endothelial cells 3.Third source: Umbilical cord: --hematopoietic, multipotency stem cells --fewer ethical limitations than human ES cells --greater longevity in the cell line generated

Markers of Differentiation 1. Markers of the mature phenotype representing terminal differentiation 2. Examples: -- Epithelium: cytokeratins -- Astrocytes: glial fibrillary acidic protein -- Erythrocyte: hemoglobin -- Hepatocyte: albumin -- Keratinocytes: transglutaminase or involucrin -- Oligodendrocyte: glycerol phosphate dehydrogenase 3. Looking for the expression of differentiation marker proteins: -- RT-PCR (?): will not necessarily confirm synthesis of proteins [to distinguish between low levels and high levels of gene expression] -- cDNA Microarray (?) -- Western blot -- Immunofluorescent staining (Flowcytometry or Fluorescent microscopy) -- 2D gel (?)

Regulation of Differentiation (1) (2) (3) (4) (5)

Induction of Differentiation Cell Interaction 1.Heterotypic Cell Interaction: --is responsible for initiating and promoting differentiation --mutual interaction between cells originating different germ layers (endoderm, mesoderm, ectoderm) promote differentiation Keratinocyte Fibroblasts

Reciprocal Paracrine Interaction: for epidermal maturation Heterotypic paracrine factor fibroblasts kerotinocyte Homotypic paracrine factor Autocrine factor

2. Homotypic Cell Interaction: -- Gap junction communication: e.g., cAMP, Ca2+, diacylglycerol -- Electrical charge -- harmonizes / balances the expression of differentiation, rather than initiating its expression Induction of Differentiation Cell Interaction

Induction of Differentiation Systemic factors / Physiological inducers or Exogenous factors / Nonphysiological inducers Physiological inducers 1.Hormones: hydrocortisone / glucagon / thyroxin 2.Vitamins: retinoids (endothelium) / vitamin D3 3.Inorganic ions: calcium (keratinocyte)

Induction of Differentiation Systemic factors / Physiological inducers or Exogenous factors / Nonphysiological inducers Nonphysiological inducers 1.Planar-polar compounds: DMSO / HMBA 2.Cytotoxic drugs: genistein / mitomycin C 3.Signal transduciton modifiers: PMA

Dimethyl sulfoxide (DMSO) 1.mouse erythroleukemia / neuroblastoma / myeloma / mammary carcinoma  response to DMSO by differentiating 2. The action of DMSO is unclear !!! 3.Possible mechanisms: -- by changes in membrane fluidity -- by influence on protein kinase C or phospholipase D -- by alternations in DNA methylation or histone acetylation 4.The induction of differentiation by DMSO may be phenotypically normal

HMBA (hexamethylene bisacetamide)

(phorbol myristate acetate)

Induction of Differentiation Cell-Matrix Interaction Matrix-induced differentiation 1.liver-derived matrix material:  induced expression of the albumin gene in hepatocyte 2.Collagen:  essential for the functional expression of epithelial cells  essential for endothelial cells to mature into capillaries 3.Synthetic matrix (poly-D-lysine):  promote neurite extension in neuronal culture 4.Matrigel (laminin+collagen+proteoglycans):  endothelial cells & epithelial cells differentiate effectively 5.Modulation of growth factor activity

Neurite extension in neuronal culture

Heparan Sulfate: cell surface proteoglycan Transmembrane heparan sulfate proteo- glycans (HSPGs) may acts as low-affinity growth factor receptor 2. HSPGs may transport these growth factors to high-affinity receptors

Induction of Differentiation Cell shape (Polarity) 1.Hepatocytes: -- flattened shape -- Cuboidal or columnar shape 2.Thyroid epithelium: in a filter well assembly --lower (basal) surface: released triiodothyronine --upper (apical) surface: released thyroglobulin

Induction of Differentiation Oxygen Tension Air/Liquid Interface (ALI)

Induction of Differentiation Oxygen Tension 1.Positioning of epidermal cells at the ALI: -- fully keratinized squamous differentiation 2.Location of alveolar type II cells at the ALI: -- for optimal differentiation 3. Location of tracheal epithelium at the ALI: -- become mucus secreting 4.Positioning of tracheal epithelium at the bottom of the dish: -- become squamous 5.ALI: -- enhances gas exchange -- w/o risking free radical toxicity

Differentiation & Malignancy 1.Cancer: a failure of cells to differentiate normally 2.With increasing progressing of cancer: -- histology of a cancer indicates poorer differentiation 3.Patients with poorly differentiated tumors: -- have a lower survival rate than patients with differentiated tumors 4.Many tumors grown in tissue culture can be induced to differentiate