University of Indonesia / Jakarta

February 27th 2019

Special Topics in Cell Biology and Cell Culture

Hans-Jürgen Mägert, Anhalt University of Applied Sciences, Köthen, Germany

 Special Topics in Cell Biology and Cell Culture

1. Basics methods of of cell culture

– Prerequirements / structure of a cell culture lab
– Basic techniques of cell culture

2. Cloning of cells
– Separation of cells (MACS, FACS)
– Limited dilution
– Isolation of Adherend Cell Clones

3. Special devices / methods in cell culture

– AVISO CellCelector
– CASY – Cell Analysis System
– MUSE MiniFACS
– Xcelligence – measurement of electric resistance of cells

4. Stem cells

5. CRISPR/Cas9 system for precise genetic engineering of cells

 Stem Cells

– May differentiate into different cell types

– Ability of unlimited proliferation

– Embryonic stem cells are pluripotent (ability to differentiate into each

body cell but not into placental cells)

– May be distinguished from progenitor cells, which have just limited

ability to proliferate and differentiate

– Also adult multipotent stem cells – but compared to embryonal stem

cells less potential to proliferate

– Great potential of embryonic stem cells for therapy of large number of

different diseases (tissue reconstitution)
 Features of Embryonic Stem Cells

– Descent from inner cell mass or epiblast (part of embryoblast)

– Capable of unlimited symmetric mitosis without differentiation (long-

term-self-renewal)

– Pluripotent – may develope to cell types, which otherwise would

descent from ektoderm, mesoderm or endoderm

– Able to integrate into developing organism => may lead to generation

of a chimeric animal possessing cells with its own genome and cells
with the imported genome

– May also colonize the genital ridge and generate germ cells
 Isolation of Embryonic Stem Cells

– Primordial germ cells (progenitor cells of egg- and sperm cells)

are isolated from early aborted fetuses and are further
developed to stem cells
– Isolation from blastocysts (embryo in 64-cell state), which have
become redundant in in vitro fertilizations
– Fusion of a body cell with an enucleated egg cell developing to
a blastocyste, from which embryonal stem cells are isolated
– Method enables generation of tissue genetically identical to the
patient (therapeutic cloning) => no rejection by the patient after
transplantation
 Isolation of Stem Cells / Progenitor Cells

– Certain types of stem cells / progenitor cells exhibit (as each

cell type) a specific pattern of cell surface proteins (CD markers
- cluster of differentiation)
– For instance, CD34 is a typical marker for hematopoietic cells
– This specific CD pattern may be used for isolation of the desired
stem cell / progenitor cells
– Methods suitable for this approach include MACS and FACS
CD Markers of Hematopoietic Cells
Adult stem cells have amongst others already been isolated
from skin, bone marrow, blood, sceletal muscles, brain and
liver
Embryonic stem cells have a great potential for therapy of large
number of different diseases (tissue reconstitution) by in vivo or
in vitro approaches

Cells (somatic cells) may also be reprogrammed into pluripotent

stem cells by use of certain compounds / messengers or genetic
manipulation => results in IPSCs (induced pluripotent stem
cells) => also usable for therapeutic approaches
Important reprogramming transcription factors are

c-Myc, Klf-4, Oct-4 and Sox-2

Growth factor LIF keeps stem cells in the

undifferentiated state
 Examples for Therapeutic Use of Pluripotent Stem
Cells

Neurodegenerative Diseases

– e.g. Pelizaeus-Merzbacher disease (mutation within gene encoding a

proteolipidprotein): Defects in glia cells surrounding and electrically isolating
nerve cells => severe neurological deficits

– Brüstle and co-workers succeeded in developing glia cells from ES cells

– Transplantation experiments with myelin-deficient rats

– Already two weeks after transplantation into spinal cord progenitor cells migrated
into recipient (target) tissue looking for nerve cell appendages lacking myelin
sheath generation =>

Attachment to nerve cell appendages, maturation to oligodendrocytes and

development of dense network of myelin sheaths
 Muscle Disorders

– e.g. Duchenne muscle dystrophy (deletion within X-chromosome leads to loss of

synthesis of the important muscle protein dystrophin)

– Gussoni and co-workers isolated adult stem cells from sceletal muscles of 3-5
weeks old mice

– Injection into tail veins of mdx mice (animal model for Duchenne muscle
dystrophy)

– Nuclei of the engrafted cells had been identified in muscle tissue => development
of new muscle tissue
 Cardiac Infarction

– Irreversible loss of heart muscle cells => ingrowing of connective tissue cells into
myocard (cicatrization) => decrease of ability to contract and performance of the
heart

– Klug and co-workers succeeded in developing heart muscle cells from ES cells of
mice

– Integrated seven weeks after implantation into hearts of adult mice

 Angiogenesis (Generation of Blood Vessels)

– To maintain functional state of certain tissues after decrease or interruption of

blood flow, usually development of new blood vessels

– Weakening of reaction in old age or in the case of certain diseases such as

diabetes and increased cholesterol level

– Kalka and co-workers succeeded in identification, isolation and ex vivo-culturing /

proliferation of endothelial stem cells from blood

– In experiments with mice transection of arteries of one hind leg and infusion of the
in vitro-proliferated endothelial stem cells

– After four weeks positive effect on vascularization detectable

 Some Expamples for Recent Progress

– Heart disease: today more than 40 clinical trials with a majority

of bone marrow, Wharton‘s jelly and adipose stem cells

– Peripheral arterial disease: 30-patient clinical trial of a stem

cell-based treatment of critical limb ischemia (CLI) showed
significant clinical effect

– Liver diseases: SC therapy has been accepted as one of the

new approaches to recolononize diseased liver – several
studies reported the hepatocyte differentiation potential of
embryonic, fetal, or adult MSC (mesenchymal stem cells) but
also IPS
Thomas Zwaka was the first, who suceeded in genetically
manipulating human embryonic stem cells =>

Potential for generation of genetically manipulated humans

(See also CRISPR/Cas9 system)

2003
2004, Caution - Fake!
Caution - Fake!
Caution - Fake!
Achtung - Fake!
 Judicial Situation in Germany

– Embryo protection law: human embryos including blastocysts are not

allowed to be generated, cloned or destroyed for research purposes

– Until April 2008 only human embryonic stem cells isolated before
January 1st 2002 were allowed to be imported to Germany

– Since April 2008 human embryonic stem cells isolated before May 1st
2007 have been allowed to be imported to Germany
 Perspectivs

Stem Cells and Whole Organ Engineering

– Also reconstruction of whole organs by means of stem cells

– Organs are decellularized, e.g. by means of solutions

containing Triton X-100 and ammonium hydroxide

– Several types of cells can be considered for recellularization

purposes: SC (embryonic, fetal, and adult SC) or the patients
autologous cells

– First success in animal models

 Perspectives

– Exact control of differentiation by means of defined growth

factors and conditions of culturing

– Thereby enabling of the investigation and therapy of a large

number of diseases such as

- Diabetes

- Neurodegenerative diseases like Parkinson‘s disease

- Liver diseases and many more........

 Special Topics in Cell Biology and Cell Culture

1. Basics methods of of cell culture

– Prerequirements / structure of a cell culture lab
– Basic techniques of cell culture

2. Cloning of cells
– Separation of cells (MACS, FACS)
– Limited dilution
– Isolation of Adherend Cell Clones

3. Special devices / methods in cell culture

– AVISO CellCelector
– CASY – Cell Analysis System
– MUSE MiniFACS
– Xcelligence – measurement of electric resistance of cells

4. Stem cells

5. CRISPR/Cas9 system for precise genetic engineering of cells

CRISPR/Cas9

The novel CRISPR/Cas9 technology (Emmanuelle

Charpentier, Jennifer Doudna) enables highly specific
(targeted) manipulations of the genome

CRISPR = Clustered Regularly Interspaced Short

Palindromic Repeats
Emmanuelle Charpentier
Jennifer Doudna
 Basic Principle of the CRISPR/Cas9 System

– Streptococcus use a DNA archive (library) to destroy entering viruses

– Their genome contains short repetitive elements – the so-called

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

– In between the CRISPR are DNA sequences positioned corresponding

to DNA segments of the viruses

– These sequences serve as template for short RNA fragments leading

an endonuclease called Cas9 to the entering viruses

– If the sequence of the viral DNA matches to that of the short leading
RNA fragment (of 23 nucleotides), Cas9 cleaves at the corresponding
site, thus, fragmenting the viral DNA
 Basic Principle of the CRISPR/Cas9 System

– This natural system may be used for deletion of any sequences from
any DNA molecules

– One just has to generate a matching leader RNA (which today is not
difficult), which directs Cas9 to the desired site

– Cas9 itself as a DNA-manipulating enzyme remains unchanged – a

great advantage compared to former methods requiring a specific
enzyme for each approach

– If artificial foreign DNA is added, there is a high propability, that during

repair processes this DNA is inserted at the cleavage site

– Allows highly specific „editing“ of genomes

The CRISPR/Cas9 complex consists of two components: gRNA
and the Cas9 enzyme

gRNA (guide RNA): fusion of a crRNA and a constant tracrRNA

crRNA: CRISPR RNA. Small non-coding RNAs that are produced by

transcription and processing of CRISPR transcripts. crRNAs contain
guide sequence (complementary to an invasive nucleic acid) and a
CRISPR-repeat-derived sequence or tag at the 5′ and/or 3′ end.

tracrRNA: Trans-activating crRNA. A small non-coding RNA of Type II

CRISPR/Cas systems that base-pairs with the 3′ tag of the crRNA and
is required for Cas9 DNA targeting activity.
Formation of the CRISPR/Cas9 Complex
Naturally, gRNA (guide RNA) is formed by partial base
pairing of two different RNA strands - crRNA and a
constant tracrRNA

For purposes of genetic engineering / manipulation, both,

crRNA and tracrRNA are usually generated by
transcription of one DNA fragment with intramolecular
complementary sequences

The resulting gRNA, thus, exhibits an intramolecular

STEM region, representing the connecting sites of crRNA
and tracrRNA
(a) Naturally occurring CRISPR systems incorporate foreign DNA sequences into CRISPR arrays, which then produce
crRNAs bearing “protospacer” regions that are complementary to the foreign DNA site. crRNAs hybridize to
tracrRNAs (also encoded by the CRISPR system) and this pair of RNAs can associate with the Cas9 nuclease.
crRNA-tracrRNA:Cas9 complexes recognize and cleave foreign DNAs bearing the protospacer sequences.
(b) The most widely used engineered CRISPR-Cas system utilizes a fusion between a crRNA and part of the tracrRNA
sequence. This single gRNA complexes with Cas9 to mediate cleavage of target DNA sites that are complementary
to the 5′ 20 nt of the gRNA and that lie next to a PAM sequence.
(c) Example sequences of a crRNA-tracrRNA hybrid and a gRNA.
Principle of Manipulation of Genomes by Use of the CRISPR/Cas9 System
Example for a vector system generating a functional CRISPR/Cas9 complex
 Delivery of CRISPR/Cas9 Components
Following methods have been used for successfully introducing
CRISPR/Cas9 components into the cell:

– RNA: Direct injection; electroporation, nucleofection, lipofectamine-

mediated transfection of non-replicating plasmid DNA to transiently
express gRNAs

– Cas9: Electroporation, nucleofection, lipofectamine-mediated

transfection of non-replicating plasmid DNA to transiently express
Cas9

– Cas9/gRNA complex: Direct injection

– For most applications, transient expression of gRNAs and Cas9 is

typically sufficient to induce efficient genome editing
Examples for published successful genetic manipulations using the CRISPR/Cas9 system
Designer Babys???
Thank you very much for your attention!