Dasar-Dasar dan Aplikasi

Stem Cells

Ahmad Hamim Sadewa

Faculty of Medicine Gadjah Mada University

 Pendahuluan

- Embryo berasal dari satu sel

- Berdiferensiasi menjadi berbagai jenis sel yang
menyusun jaringan dan organ
- Sel tersebut dikenal sebagai stem cell (sel
punca)
 Basic Principles of Stem Cells

Stem cells
retain the ability to renew themselves through
mitotic cell division
can differentiate into adverse range of
specialized cells types (plasticity)

unspecialized cells

potential as therapeutic agents

(leukemia, heart disease, Parkinson, Hirschsprung Disease etc.)
Retain the ability to renew themselves

Stem cells have a special ability to divide and renew

themselves for extended periods of time.
Cells such as muscle or nerve don't normally replicate,
but stem cells will repeatedly proliferate.
An initial population of stem cells can produce millions
of cells within several months in a laboratory setting.
Embryonic stem cells appear to be able to proliferate
for a year or even longer without ever developing into
specialised cells.
The exact mechanisms still under investigation.
Can differentiate into specialized cells types

Stem cells have many genes regulate the internal

signals that trigger this process.
These genes carry the specific code, or instructions,
for all the parts and functions of a cell.
Currently, scientists are searching for similarities and
differences between the signals from one stem cell to
another. These answers will allow to find ways to control
stem cell differentiation.
Unspecialized cells
Stem cells lack the specific parts that allow them to
perform specialised functions in the body.
A stem call does not have a specialised function but it has
the capacity to differentiate into a specialised cell that can
carry out these functions.
Research has allowed scientists to grow stem cells in the
laboratory, but the precise signals that trigger a stem cell
to proliferate are still largely unknown. Once this is
determined, scientists will be able to grow stem cells in
the laboratory with greater success.
 History
1968: the first bone marrow transplant was performed to successfully
treat two siblings with severe combined immunodeficiency
1978: Stem cells were discovered in human cord blood
1981: First in vitro stem cell line developed from mice
1988: Embryonic stem cell lines created from a hamster
1995: First embryonic stem cell line derived from a primate
1997: Cloned lamb from stem cells
1997: Leukaemia origin found as haematopoietic stem cell, indicating
possible proof of cancer stem cells
1998 : Isolation cells from the inner cell mass of early embryos and
developed the first embryonic stem cell lines.
1999 and 2000: manipulation adult mouse tissues could produce
different cell types
 Sources of Stem Cells

Embryonic stem cells: they are extracted from embryos

and are thought to hold the most potential, because these
cells can give rise to virtually any specialised cell in the
human body.

Adult stem cells: these are present in adult tissues such

as the bone marrow, brain and blood but are limited in
potential relative to embryonic stem cells.

Cord blood stem cells: this source of stem cells is derived

from cord blood and is thought to hold enormous potential
in treating disease.
 Potency of Stem Cells

Totipotent stem cells: they can differentiate into any type of

cell in the human body, including the placenta.

Pluripotent stem cells: they descend from totipotent stem

cells and after several days, can differentiate into any type
of cell except for totipotent stem cells.

Multipotent stem cells: these descend from pluripotent stem

cells and can differentiate into many cell lines within a
specific type of tissue.

Unipotent stem cells: this type of stem cells is a

descendant of a multipotent stem cell and can give rise to a
single cell type.
 Totipotent
Pluripotent
Multipotent Multipotent
Stem Cells
Pluripotent Stem Cells
Pluripotent stem cells are often termed 'true' stem cells
because they have the potential to differentiate into
almost any cell in the body.
After fertilization, a single cell results. This cell - the
fertilised egg that is totipotent - has the potential to create
an entire organism. In the initial hours and days following
fertilisation, this single totipotent cell divides into more
totipotent cells that are exact copies of the original.
Approximately four days after fertilisation, the totipotent
cells start to specialise and form a cluster of cells known
as a blastocyst.
The blastocyst has yet another smaller group of cells
known as the inner cell mass and it is these inner
pluripotent stem cells that will go on to create most of the
cells and tissues in the human body.
These pluripotent stem cells are therefore different than
totipotent stem cells because they don't develop into a
complete organism.
A pluripotent cell won't give rise to the placenta or other
tissues that are vital for foetal development. It will still
develop into the other specialised cell types in the
human body, such as nerve or heart cells.
 Types of Pluripotent Stem Cells

Embryonic stem cells are isolated from the inner cell mass
of the blastocyst. The embryos are excess ones produced
from in vitro fertilisation, but the practice is still controversial
because it does destroy the embryo, which could have
been implanted to create a baby.

Embryonic germ cells are taken from aborted foetuses and

these pluripotent cells are derived from very early cells.
These early cells are those that can become sperm and
eggs.

Embryonic carcinoma or cancer cells are isolated from a

type of tumour that sometimes occurs in a foetus.
Multipotent Stem Cells
A multipotent stem cell can give rise to other types of
cells but it is limited in its ability to differentiate.
Examples of multipotent stem cells include those in the
brain that give rise to different neural cells and glia or
haematopoietic cells, which can give rise to different
blood cell types, but they can't create brain cells.
A multipotent stem cell known as a mesenchymal stem
cell can give rise to several cell types. This particular
stem cell has been found to give rise to bone, muscle,
cartilage, fat, and other similar tissues.
Unipotent Stem Cells

A unipotent stem cell refers to a cell that can differentiate

along only one lineage.
The unipotent adult stem cells in the body's tissues will
only give rise to one cell type, but they do still have the
important property of self-renewal that is shared by all
stem cells.
Despite their differentiation potential being limited,
unipotent cells still have vast therapeutic potential to
treat injuries and diseases.
Stem Cell Niche

• Stem cell niche is the microenvironment in which stem

cells are found, which interacts with stem cells to
regulate stem cell fate, in vivo or in vitro.

• During embryonic development, various niche factors act

on embryonic stem cells to alter gene expression, and
induce their proliferation or differentiation for the
development of the fetus.

• The stem cells and niche may induce each other during
development and reciprocally signal to maintain each
other during adulthood.
Several factors are important to regulate stem cell
characteristics within the niche:

• cell-cell interactions between stem cells

• interactons between stem cells and neighbouring
differentiated cells
• interactions between stem cells and adhesion molecules
• extracellular matrix components
• the oxygen tension
• growth factors (klf4, oct3/4, sox2, c-myc)
• cytokines
• physiochemical nature of the environment including the
pH, ionic strength (eg calcium concentration, metabolites
like ATP are also important.
 Vertebrate Adult Stem Cell Niches

A. Hematopoietic stem cell niche

Vertebrate hematopoietic stem cells niche is formed by
cells subendoosteal osteoblasts, sinusoidal endothelial
cells and bone marrow stromal (also sometimes called
reticular) cells which includes a mix of fibroblastoid,
monocytic and adipocytic cells.
B. Hair follicle stem cell niche
The bulge area at the junction of arrectores pili muscle to
the hair follicle sheath has been shown to host the skin
stem cells with maximum span of developmental
potential.
C. Intestinal stem cell niche
The subepithelial fibroblast/myofibroblast network which
surround the intestinal crypts constitute the niche.
 Diferensiasi

Stem cell dapat tumbuh dan berkembang menjadi

sel dewasa dengan fungsi spesifik
1. Stem cell hematopoeitik : seluruh jenis sel
darah seperti eritrosit dan leukosit
2. Stem cell jaringan saraf : astrosit,
oligodendrosit dan neuron
3. Stem cell jaringan kulit : sel penyusun
epidermis dan keratinosit
4. Stem cell mesenkhimal : osteosit, kondrosit,
adiposit, sel penyusun jaringan ikat
5. Stem cell jantung : endotel, kardiomiosit
 Transdiferensiasi

Adalah diferensiasi di luar alur diferensiasi yang

biasanya terjadi
1. Stem cell mesenkhimal menjadi sel saraf
2. Stem cell hematopoeitik menjadi kardiomiosit
3. Stem cell hepar menjadi sel beta-pankreas
 Stem Cell Hasil Induksi
Adalah stem cell yang berasal dari sel dewasa
(fibroblas) yang diinduksi menjadi sel pluripoten
sehingga serupa dengan stem cell embrionik
dengan menyisipkan faktor transkripsi (iPS =
induced pluripotent stem cell) (Takahashi dan
Yamanaka, 2006)
Mempunyai banyak kelemahan seperti efisiensi
penyisipan faktor transkripsi rendah,
penggunaan virus, efek samping dan kemungkin
sifat onkogenik/karsinogenik.
 Mekanisme Regenerasi Jaringan oleh
Stem Cell

1. Diferensiasi oleh stem cell

- Kelainan darah dengan stem cell
hematopoeitik
- Parkinson, stroke dengan stem cell neural
- Kelainan tulang dan otot dengan stem cell
saraf
2. Produksi faktor pertumbuhan
- Stem cell mampu bertindak sebagai sel tropik
yang memproduksi sitokin dan faktor
pertumbuhan seperti interleukin, stimulating
factor dll
Problems

Immune rejection

Immunosupressive drugs
Nuclear transfer
Using adult stem cells

Contamination

Certain biological activities

Identification and propagation stem cells within an

actual tissue culture in a laboratory setting
Ethical Issues
Over the last few years, national policies and
debate amongst the public as well as religious
groups, government officials and scientists have led
to various laws and procedures regarding stem cell
harvesting, development and treatment for research
or disease purposes.

Fabrication

This was the case in 2004 to 2005, when Hwang

Woo-Suk, a Korean researcher, claimed to have
produced human embryonic stem cell lines from
unfertilised human eggs
Aplikasi Stem Cell pada Hirschsprung Disease

Hirschsprung Disease

kelainan bawaan dengan insidensi 1 : 5000 kelahiran hidup

pria : wanita = 4 : 1
di RS Sardjito 40 – 60 kasus per tahun, berbagai etnik

Tidak terdapat plexus submukosa dan myenterikus

(aganglion)
usus aganglion spasme, proksimal aganglion hipertrofi

mortalitas bila tidak ditangani 80%, bila ditangani 30%

Diagnosis HD
Anamnesis
keterlambatan mekoneum 24 – 48 jam
perut kembung, konstipasi berulang
muntah warna hijau/fekal
Pemeriksaan Fisik
distensi abdomen
Rectal toucher : mencengkeram, bila
dilepas feses menyemprot
Pemeriksaan Penunjang
biopsi
radiologi : barium enema (panjang segmen aganglion)
Komplikasi
obstruksi intestinal
konstipasi
enterokolitis
malnutrisi

Manajemen
dekompresi (rectal tube dan NGT)
stabilitas cairan, elektrolit, asam basa dan temperatur
keseimbangan nutrisi
kolostomi
operasi definitif
 Patofisiologi
Normal : Sel neuroenterik bermigrasi dari krista neuralis
dari oral ke anal; sampai esofagus minggu V, midgut
minggu VII, colon distal minggu XII

kegagalan migrasi Faktor genetik

RET (rearranged
gagal maturasi during transfection)
degradasi/destruksi GDNF
EDNRB

HSCR
 ENS Origins and Development

Enteric Nervous System (ENS) derives from migration of

neural crest cells

When neural crest cells enter the foregut, they are

termed enteric neural crest-derived cells (ENCCs)
which are ENS progenitors

ENCCs proliferate actively and generate enteric neurons

(ENS) and glia that are present in adult intestine
Myentericus and submucosal plexus
Gene Expression of Cells Contributing to ENS
Vagal neural crest invade
anterior foregut and
midrate from rostral to
caudal

At embyonic day 8.5-9 in

the mouse
First, SOX10 and EDNRB
were expressed
Upon entering the foregut at E9-9.5, ENCCs begins
to express RET, GDNF (green), EDN3 (pink)

GDNF
EDN3
 GDNF and EDN3

At E11, GDNF and EDN3 were expressed in high

level at caecum area
Cells undergoing neural differentiation express RET
and TUJ1, undergoing glial cell differentiation express
SOX10 and GFAP4
ENS Phenotype
Signaling pathway of RET, EDNRB, EDN3 and GDNF
Nature Review Neuroscience, June 2007
Hirschprung Disease Genes and Loci
ENS Stem Cells : Isolation and transplantation
Differentiation
Isolation
Transplantation

GDNF
addition
enhance
migration
Key Elements of Stem Cells Therapy for HSCR

enteric precursor cells can be isolated from tissue

taken from patient with HSCR
culture conditions exist to maintain enteric precursor in
a progenitor state
aganglionic gut regions are capable of supporting at
least some aspects of ENS development

preliminary evidence suggests that transplanted cells

can contribute to some aspects of ENS function
Questions

Who is eligible to be transplanted?

What kind of stem cells should be transplanted?

When stem cells should be transplanted?

How to monitor side effects?

 Conclusion

Stem cell therapy for many diseases is

significantly improve at molecular and technical
level towards application in human.
Some technical difficulties combined with ethical
problems should be overcome.