IDENTIFICATION and CHARACTERISATION of TISSUE-SPECIFIC STEM CELLS
Ontogeny of Adult Stem Cells
Ontogeny or ontogenesis is the entire sequence of events involved in the
development of an individual organism.
Prospective vs Retrospective Isolation
Prospective isolation- looking ahead. Stem cells are isolated by
physical properties before they differentiate.
Stem cell markers: genes and protein products used to identify
stem cells.
Retrospective isolation- looking backwards. When cells
behaviour is examined, leading to the retrospective identification of the
presence of a stem cell.
Functional assays: assays designed to evaluate aspects of stem cell behaviour, in particular self-
renewal, and differentiation.
IN-VITRO ASSAYS:
Colony-Forming Efficiency Assay
In vitro functional assay.
They evaluate self-renewal potential.
Cells are plated at low density, to permit clonal analysis.
Experiment: colony-forming efficiency of ocular keratinocytes. Three clonal types of keratinocytes
with different capacities for multiplication (Y. Barrandon and H. Green). When primary cultures of
normal cells are cloned, three types of colony grow.
Holoclones- believed to be derived from stem cells. Started at single cells and give rise to colony.
Meroclones- from transit- amplifying cells. Close to stem cells. Don’t contain as many cells.
Paraclones- from differentiated cells. Give rise to some progeny. No large dense
colony.
Experiment: determination of the number of cell doubling generated by
conjunctival clones. Ocular keratinocytes were serially cultivated until they
reached senescence (process which a cell ages and permanently stops dividing but
doesn’t die). They cultured cells from the eye and they observed which types of
colonies they give rise to. This is a retrospective assay, no biomarker is used.
These colony forming assays can be preceded by flow cytometry purification of
cell populations. Taking whole population of cells and use different ways of
labelling them (antibodies- tags) and run the cells through detector. It tells which
genes or proteins are expressed in the cells. Then these group of cells are taken to
functional assays to be analysed. (prospective).
Sphere-Forming Assays
In vitro functional assay.
3D assay.
Capacity to evaluate self-renewal and differentiation at the single cell
level in vitro.
Detection of epitopes using antibodies.
GFAP-GFP is a cytoplasmic marker.
, Growth of Organoids (In Vitro
Functional Assay)
An organoid is a 3D organ-bud grown in
vitro.
Reconstitutes aspects of the tissue
microanatomy and (ideally) physiology.
Organoids are used to model different
aspects of developmental and cell
biology.
Stem cell lineage selection choices.
IN-VIVO FUNCTIONAL ASSAYS:
Label Retention Assay
All cells within a living tissue (white circles, left) are
labelled during the pulse period (blue cells).
During the chase period, labelling stops and the cells
dilute the dye according to their proliferation rate.
Quiescent cells retain the dye, which can still be
detected at the end of the chase period.
Kabel retention assays permit evaluation of the
proliferative history of a cell.
Braun et al. BrdU label retaining cells in skin.
Identifies slow cycling population. Located in bulge region.
Tumbar et al. .Genetic labelling such as histone 2B-green fluorescent protein (H2B-GFP).
Cell Lineage Tracing Assay (Fate Mapping)
Lineage tracing is a method to study stem cell behaviour in vivo.
Used to elucidate the role of stem cells in tissue development, homeostasis and disease.
Permanently (genetically) label a particular cell type (e.g. using Cre-lox recombination).
Establishes the relationship between a cell at one stage of development, and its progeny at later stages
of development. Look at later times to see if:
- Cell has self-renewed (number of labelled cells per clone has increased).
- Cell has undergone multi-lineage differentiation (clones contain multiple cell types).
Ontogeny of Adult Stem Cells
Ontogeny or ontogenesis is the entire sequence of events involved in the
development of an individual organism.
Prospective vs Retrospective Isolation
Prospective isolation- looking ahead. Stem cells are isolated by
physical properties before they differentiate.
Stem cell markers: genes and protein products used to identify
stem cells.
Retrospective isolation- looking backwards. When cells
behaviour is examined, leading to the retrospective identification of the
presence of a stem cell.
Functional assays: assays designed to evaluate aspects of stem cell behaviour, in particular self-
renewal, and differentiation.
IN-VITRO ASSAYS:
Colony-Forming Efficiency Assay
In vitro functional assay.
They evaluate self-renewal potential.
Cells are plated at low density, to permit clonal analysis.
Experiment: colony-forming efficiency of ocular keratinocytes. Three clonal types of keratinocytes
with different capacities for multiplication (Y. Barrandon and H. Green). When primary cultures of
normal cells are cloned, three types of colony grow.
Holoclones- believed to be derived from stem cells. Started at single cells and give rise to colony.
Meroclones- from transit- amplifying cells. Close to stem cells. Don’t contain as many cells.
Paraclones- from differentiated cells. Give rise to some progeny. No large dense
colony.
Experiment: determination of the number of cell doubling generated by
conjunctival clones. Ocular keratinocytes were serially cultivated until they
reached senescence (process which a cell ages and permanently stops dividing but
doesn’t die). They cultured cells from the eye and they observed which types of
colonies they give rise to. This is a retrospective assay, no biomarker is used.
These colony forming assays can be preceded by flow cytometry purification of
cell populations. Taking whole population of cells and use different ways of
labelling them (antibodies- tags) and run the cells through detector. It tells which
genes or proteins are expressed in the cells. Then these group of cells are taken to
functional assays to be analysed. (prospective).
Sphere-Forming Assays
In vitro functional assay.
3D assay.
Capacity to evaluate self-renewal and differentiation at the single cell
level in vitro.
Detection of epitopes using antibodies.
GFAP-GFP is a cytoplasmic marker.
, Growth of Organoids (In Vitro
Functional Assay)
An organoid is a 3D organ-bud grown in
vitro.
Reconstitutes aspects of the tissue
microanatomy and (ideally) physiology.
Organoids are used to model different
aspects of developmental and cell
biology.
Stem cell lineage selection choices.
IN-VIVO FUNCTIONAL ASSAYS:
Label Retention Assay
All cells within a living tissue (white circles, left) are
labelled during the pulse period (blue cells).
During the chase period, labelling stops and the cells
dilute the dye according to their proliferation rate.
Quiescent cells retain the dye, which can still be
detected at the end of the chase period.
Kabel retention assays permit evaluation of the
proliferative history of a cell.
Braun et al. BrdU label retaining cells in skin.
Identifies slow cycling population. Located in bulge region.
Tumbar et al. .Genetic labelling such as histone 2B-green fluorescent protein (H2B-GFP).
Cell Lineage Tracing Assay (Fate Mapping)
Lineage tracing is a method to study stem cell behaviour in vivo.
Used to elucidate the role of stem cells in tissue development, homeostasis and disease.
Permanently (genetically) label a particular cell type (e.g. using Cre-lox recombination).
Establishes the relationship between a cell at one stage of development, and its progeny at later stages
of development. Look at later times to see if:
- Cell has self-renewed (number of labelled cells per clone has increased).
- Cell has undergone multi-lineage differentiation (clones contain multiple cell types).
