LE 8 – Organoid Technology and
applications
December 8 t h , 2023
Pre-clinical cancer research tools: cell lines and animal models
Pros
▪ Easy to culture and expand
▪ Cheap
▪ Easy to manipulate (CRISPR)
▪ Many commercially available cell lines for many cancers
Cons
▪ After years of growing in plastic, the cells no
longer resemble primary cells of origin
▪ Karyotype is a mess
▪ Grown in 2D or in suspension
▪ Absence of stroma
Karyotype of HeLa cells assessed by multicolor
fluorescence in situ hybridization (M-FISH).
Animal models
Pros
▪ Relatively easy to culture and manipulated (CRISPR)
▪ Can be used to study cancer initiation and development in a live organism (patient derived
xenografts)
Cons
▪ Expensive
▪ Millions or lab animals are sacrificed across the globe each year
▪ Experiments take a long time
▪ Human biology is not the same as mouse, rat, or zebrafish biology
Until 10 years ago, the majority of preclinical cancer research was done using cancer cell lines and
animal models. This is mainly because growing primary human (cancer) cells in a culture dish is
extremely challenging.
,>200 different terminally differentiated cell types in the human body
Embryonic versus adult stem cells
Embryonic stem cells
▪ Pluripotent: able to mature into any cell type of the body
o Embryonic stem cells appear at the blastocyst stage of the embryo
Adult Stem Cells
▪ Multipotent: can mature into a few cell types
▪ Unipotent: can mature into one cell type
o All organs have one or more adult stem cells
o Maintain and repair tissue
Champion adult stem cell is believed to reside in the intestine: every day 300 grams of new cells are
produced in a human adult intestine. For many years, the exact identity of this intestinal adult stem cell
remains elusive.
, Crypt and villi structure of the intestinal epithelium
Identification of Lgr5 as the adult stem cell marker in the intestine
Crypts were believed to contain stem cells; villi contain differentiated cells. Epithelial self-renewal is
driven by Wnt signaling. Lgr5 is one of 80 genes in the intestine that becomes activated upon Wnt
stimulation. Marking Lgr5 cells in the intestine with GFP revealed that this protein marks adult stem
cells. Lgr5 stem cells were also identified in other tissues. This allows isolation and in vitro culture of
these cells (although this still turned out to be very difficult).
Epithelial self-renewal in the intestine
Single isolated Lgr5+ stem cells can grow out to form ‘mini guts’ or organoids
Isolated Lgr5 cells are embedded in semi-solid culture medium and a cocktail of growth factors. Cells
from cystic structures and eventually organoids form, which harbor a crypt and villus domain and a
central cavity in which dead cells are shed. Intestinal organoids contain all the cell types that are known
to exist in the intestinal epithelium. Organoids can
be maintained, propagated and split for a long
period of time. Allows to study adult stem cell
homeostasis and differentiation in vitro. Intestinal
cancer originates from mutation in Lgr5 cancer
stem cells.
applications
December 8 t h , 2023
Pre-clinical cancer research tools: cell lines and animal models
Pros
▪ Easy to culture and expand
▪ Cheap
▪ Easy to manipulate (CRISPR)
▪ Many commercially available cell lines for many cancers
Cons
▪ After years of growing in plastic, the cells no
longer resemble primary cells of origin
▪ Karyotype is a mess
▪ Grown in 2D or in suspension
▪ Absence of stroma
Karyotype of HeLa cells assessed by multicolor
fluorescence in situ hybridization (M-FISH).
Animal models
Pros
▪ Relatively easy to culture and manipulated (CRISPR)
▪ Can be used to study cancer initiation and development in a live organism (patient derived
xenografts)
Cons
▪ Expensive
▪ Millions or lab animals are sacrificed across the globe each year
▪ Experiments take a long time
▪ Human biology is not the same as mouse, rat, or zebrafish biology
Until 10 years ago, the majority of preclinical cancer research was done using cancer cell lines and
animal models. This is mainly because growing primary human (cancer) cells in a culture dish is
extremely challenging.
,>200 different terminally differentiated cell types in the human body
Embryonic versus adult stem cells
Embryonic stem cells
▪ Pluripotent: able to mature into any cell type of the body
o Embryonic stem cells appear at the blastocyst stage of the embryo
Adult Stem Cells
▪ Multipotent: can mature into a few cell types
▪ Unipotent: can mature into one cell type
o All organs have one or more adult stem cells
o Maintain and repair tissue
Champion adult stem cell is believed to reside in the intestine: every day 300 grams of new cells are
produced in a human adult intestine. For many years, the exact identity of this intestinal adult stem cell
remains elusive.
, Crypt and villi structure of the intestinal epithelium
Identification of Lgr5 as the adult stem cell marker in the intestine
Crypts were believed to contain stem cells; villi contain differentiated cells. Epithelial self-renewal is
driven by Wnt signaling. Lgr5 is one of 80 genes in the intestine that becomes activated upon Wnt
stimulation. Marking Lgr5 cells in the intestine with GFP revealed that this protein marks adult stem
cells. Lgr5 stem cells were also identified in other tissues. This allows isolation and in vitro culture of
these cells (although this still turned out to be very difficult).
Epithelial self-renewal in the intestine
Single isolated Lgr5+ stem cells can grow out to form ‘mini guts’ or organoids
Isolated Lgr5 cells are embedded in semi-solid culture medium and a cocktail of growth factors. Cells
from cystic structures and eventually organoids form, which harbor a crypt and villus domain and a
central cavity in which dead cells are shed. Intestinal organoids contain all the cell types that are known
to exist in the intestinal epithelium. Organoids can
be maintained, propagated and split for a long
period of time. Allows to study adult stem cell
homeostasis and differentiation in vitro. Intestinal
cancer originates from mutation in Lgr5 cancer
stem cells.
