Biotechnological applications

of induced human stem cells

Interdepartmental Postgraduate Program of Studies

Biotechnology

14th April 2022

Institute of Applied Biosciences/Centre for Research
and Technology Hellas

Spyros Petrakis, PhD

spetrak@certh.gr
www.inab.certh.gr
 Molecular Neurobiology lab

Aim: Develop 2D/3D models of neurodegenerative diseases

using biotechnology techniques
Study the molecular mechanisms of neurodegeneration
Identify chemical compounds that could be used for drug development
 Definition: Biotechnology is a broad area of biology
involving the use of living systems and organisms to develop
or make products. www.wikipedia.com

Various (colors) sectors exist based on their applications

Medical biotechnology is the use of biotechnology in the medical

and pharmaceutical industries, and health preservation. This
branch involves the production of vaccines and antibiotics,
regenerative therapies, creation of artificial organs and new
diagnostics of diseases. As well as the development of
hormones, stem cells, antibodies, siRNA and diagnostic tests.
www.wikipedia.com

Organoid/mini brain
 Bioethics, biosafety and bioeconomy
of biotechnological products

Bioethics is the study of the ethical issues emerging from

advances in biology and medicine.
www.wikipedia.com
Example: Genome editing in gene therapies or experimental
use of animals.

Biosafety is the prevention of large-scale loss of biological

integrity, focusing both on ecology and human health.
www.wikipedia.com
Example: Safety of vaccines for COVID-19.

Bioeconomy refers to economic activity involving the use of

biotechnology and biomass in the production of goods,
services, or energy.
www.wikipedia.com
Example: Commercial use of cell therapies (e.g. SMA)
 Neurodegenerative diseases

Proteotoxicity, aggregation and selective neuronal cell death

Phenotype is associated with mutations in toxic proteins
 DNA expansion mutations

Huntington’s disease: huntingtin

Spinocerebellar ataxias: ataxins
Earlier onset with longer
mutations
 DNA expansion mutations

Ataxin-1 isoforms

ATXN1/DAPI ATXN1/DAPI

Wild-type mouse SCA1 Tg mouse WT SCA1

 Mechanisms of diseases
polyQ
polyQ polymers
polyQ oligomers
monomers
Aging

Apoptosis Necrosis
Commonalities among neurodegenerative diseases

Haenig et al., Cell Reports, 2020.

 Precision medicine

Precision medicine is a medical model that proposes the customization of

healthcare, with medical decisions, treatments, practices, or products being
tailored to a subgroup of patients, instead of a one drug fits all model.
www.wikipedia.com
 Precision medicine

Response to drugs is affected by our

genetic profile.

Activity of enzymes, mutations in related

genes or other risk factors.

Molecular diagnostics to identify

Individual differences

Polymerase chain reaction

Next-generation sequencing
 Precision medicine in Greece

Precision medicine in Oncology

Precision Medicine in Cardiology
Precision Medicine in Neurodegenerative Diseases

https://oncopmnet.gr
https://inab.certh.gr/precision-medicine
 Precision medicine in Greece

Precision medicine in Oncology

Analysis of 38+ genes from easily accessible cancer cells
(peripheral blood or solid tumor cells)

Precision Medicine in Cardiology

Analysis of cardio-vascular system cells

Precision Medicine in Neurodegenerative Diseases

Not easily accessible cells

Need for neuronal cells that can be expanded and analyzed in the lab
 Stem cells

Embryo

Terminally differentiated cells Pool of undifferentiated stem

cells

Patient neural stem cells

Neural stem cells
Healthy neural stem cells

Problems in their use?

 Genetic engineering

Genetic engineering, also called genetic modification or genetic

manipulation, is the direct manipulation of an organism's genes using
biotechnology.

It involves the use of Recombinant DNA (rDNA), DNA molecules by

laboratory methods of genetic recombination that bring together genetic
material from multiple sources, creating sequences that would not
otherwise be found in the genome.
www.wikipedia.com
 Fluorescent biobricks

Green Fluorescent Protein (GFP)

Osamu Shimomura
from Aequorea victoria

The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in
Chemistry for 2008 jointly to
Osamu Shimomura, Marine Biological Laboratory (MBL), Woods Hole, MA, USA and
Boston University Medical School, MA, USA,
Martin Chalfie, Columbia University, New York, NY, USA
and
Roger Y. Tsien, Howard Hughes Medical Institute, University of California, San Diego, La
Jolla, CA, USA
for the discovery and development of the green fluorescent protein, GFP”.
 Fluorescent biobricks

Variants of GFP (from blue to red color)

Indicative application: differential labeling of neurons with fluorescent proteins

Brain hippocampus involved in emotions, learning and memory formation

Can you think of a result of this experiment?
 Synthetic biology tools

Transposon-mediated genetic modification

•Transposons represent an alternative to viruses for
delivery of genes into cells.
•Their advantages include lack of antigenicity, low
cytotoxicity.
• They prefer TA-rich intronic sequences for insertion
minimizing the risk of oncogenesis.

More tools
Inducible systems. “Manual” regulation of gene expression

Tet-On system
 Synthetic cells

Cells expressing a pathogenic protein causing neurodegeneration (ATXN1Q82)

in fusion with GFP using the inducible Tet-On system

Measurement of fluorescent cells Detection of recombinant protein

(ATXN1Q82) using antibodies
 Synthetic cells

YFP-ATXN1Q82

+Dox 2d +Dox 5d +Dox 10d

YFP-ATXN1Q30

+Dox 2d +Dox 5d +Dox 10d

Deposition of protein aggregates.

What is their effect in cells?
 Synthetic cells

Microscopical cell imaging/measurement of aggregate size

-Dox +Dox 2D +Dox 5D +Dox 10D

Which molecule localizes in the nucleus of a cell?

 Synthetic cells

Measurement of cellular parameters

More aggregates-more oxidative stress More aggregates-higher proteasomal activity

Does this makes sense?
 Molecular diagnostics (DNA sequencing)
DNA fragmentation, modification and attachment in cartridges

PCR amplification of billions of fragments

Addition of fluorescent free nucleotides

 Molecular diagnostics (DNA sequencing)

Masive parallel next generation sequencing (NGS)

which is completed in 48 hrs with an affordable cost
Animation of DNA sequencing
https://www.youtube.com/watch?v=v10bUR2aL5g
Molecular diagnostics (DNA sequencing)

Computational assembly of the whole DNA

sequence (billions of bp) using 75 bp long
reads/fragments and a reference genome.

Each nucleotide is read several times

 Synthetic cells
Sequencing
D2 D10
D0 D0

Visualization of gene expression

Which protein machineries are

affected by protein aggregation?

Network analysis.
(e.g. oxidative stress)

How would you proceed in order

to treat the disease?
Synthetic cells
 Different cell types carrying the same DNA

All cell types arise from one cell and carry the same DNA sequence

How can cells with the same DNA have so many different shapes?
 Cellular differentiation

Differentiation is closely related to transcription regulation

Transcription regulation is mediated

by transcription factors that bind to
specific DNA sequences

Animation of transcription regulation

https://www.youtube.com/watch?v=MkUgkDLp2iE
 Cellular differentiation

Transcription factors regulate gene expression and drive cellular morphology

Activation of genes is responsible for the shape of the body

Specific transcription factors are present in specific areas of the embryo

and commit cells to a specific type
 Stem cells

Embryo

Terminally differentiated cells Pool of undifferentiated stem

cells

Induced pluripotent stem cells Neural stem cells

 Engineered neurons

Obtained by written
consent form defining
their use

Transcription factors
Proteins that control the expression pattern of genes

Genetic modification with:

Somatic cells
1. Integrating viruses
iPSCs 2. Non-integrating (episomal)
vectors
Which ones would you choose?
 Cellular products

Any type of (stem) cell

Disease modeling: Compare cells from AD patients and healthy individuals

Drug discovery: Improvement of a disease phenotype

Precision medicine: Testing and selection of an appropriate treatment

Personalized medicine

Cell therapy: Genetic correction and transplantation for regenerative purposes

 Cellular products
Disease modeling

Commercially available stem cell lines

https://www.axolbio.com
 Cellular products
Disease modeling

Commercially available stem cell lines

https://www.axolbio.com
 Cellular products
Disease modeling

Bioethics: Is there clear consent form for the manipulation, handling and
commercialization of the biological material? Are personal data protected
according to GDPR?

Biosafety: No risks as long as they are used for experimental purposes

Bioeconomy: More expensive than common laboratory cell lines, expensive

media but cost-effective culturing and characterization
 iPSC-based models

iPSCs are partially rejuvenated and are not appropriate

for modeling of age-related diseases

Induced neural stem cells

Induced neurons
Induced neural stem cells
 Induced neural stem cells

Oligodendrocyte differentiation protocol

 Induced neural stem cells

Electrophysiological activity, Integration into animal brains

 Cellular products
Cell therapy

Regenerative therapy
Non-integrating
viruses

Tissue-affected
cell type
CRISPR-mediated
genetic correction

Autologous vs heterologous transplantation?

 CRISPR-mediated genetic correction

Bacterial defense system for protection from viruses.

Adapted to serve targeted genetic engineering

Animation of genome editing with CRISPR

https://www.youtube.com/watch?v=2pp17E4E-O8
 Cellular products
Cell therapy

FDA approves first test of CRISPR to correct genetic defect

causing sickle cell disease
(https://news.berkeley.edu/2021/03/30/fda-approves-first-test-of-crispr-to-correct-genetic-defect-causing-
sickle-cell-disease/)

Bioethics: Are all personal data protected according to GDPR?

Biosafety: Genetic correction should not cause mutagenesis or carcinogenicity.

Cell expansion in media for clinical use. Clinical testing.

Bioeconomy: Expensive treatments (several millions per person).

 Cellular products
Cell therapy

FDA approves first test of CRISPR to correct genetic defect

causing sickle cell disease
(https://news.berkeley.edu/2021/03/30/fda-approves-first-test-of-crispr-to-correct-genetic-defect-causing-
sickle-cell-disease/)

(20-100) (50-300) (300-3.000) Pharmacovigilance

 Failure of genetic therapies-the case of Huntington’s disease

http://uniqure.com/gene-therapy/huntingtons-disease.php https://www.nature.com/articles/d41586-021-01177-7

Causes of failure (?)

Adverse or no significant effect of the virus

Inability of the virus to target disease cells

Integration into e.g. introns and reduced expression of the transgene

Cross-reactivity with other genes (off-target effect)

 Cellular products
Cell therapy

Transplantation of green
stem cells in rat brain

Problems
Limited integration into the host tissue

Limited viability and apoptosis of cells

Lack of 3D structure resembling the affected area

 Cellular products
Cell therapy

Solutions:
1. Engineered scaffolds using solid or biodegradable materials

2. Bioprinting using hydrogel-based materials

Generate 3D human neural tissue

Cellular products
Engineered scaffolds
smNPCs viability
0.8

0.6

0.4

0.2

0
Day 1 Day 10 Day 15 Day 23

Neural stem cells

Neurons
 Cellular products
Bioprinted implants
Neurons grown on biocompatible materials

Organoid/mini brain
3D printed AD brain (?)
 Brain organoids

https://mindresearchfacility.nl/

Can you think of bioethics, biosafety and bioeconomy issues?

Please contact me at spetrak@certh.gr for any inquiries