Fish stem cells and their applications by B

Fish stem cells and their applications
By:
BHUKYA BHASKAR
FISHERIES
Zebra fish produce Sunscreen called
gadusol, protects the growing embryo
from harmful UV-Rays. Similar to that of
sheild for protecting stemcells from
harmful UV-Rays
Tilapia is an important farmed freshwater
species of fish that has become one of the
most popular sources of collagen for bone
tissue engineering.

Introduction
• Stem cells are a class of undifferentiated cells, have the potential to reproduce
themselves by mitotic cell division, generate progeny destined to differentiate into
functional cell types, persist for a long time and its behaviour is regulated by the
micro environment
• Stem cells are cells with the potential to develop into many different types of cells
in the body.
• They serve as a repair system for the body.
• There are two main types of stem cells: embryonic stem cells and adult stem cells.
• What are 3 types of stem cells?
• Hematopoietic Stem Cells (Blood Stem Cells) Mesenchymal Stem Cells. Neural Stem
Cells.
• Types of stem cells
• Hemopoietic stem cell (HSCs)
• Mesenchymal stem cell (MSCs)
• Neural stem cell (NSCs)
• Embryonic stem cell (ESCs)
• Induced pluripotent stem cell (iPSCs)
• Where are stem cells found?
• Adult stem cells have been found in most parts of the body, including brain, bone
marrow, blood vessels, skin, teeth and heart. There are typically a small number of
stem cells in each tissue.

Cont...
• Two defining characteristics of a stem cell are self-renewal by mitotic
division and the ability to differentiate into a specialized adult cell type
(Çek et al., 2016).
• Four properties of stem cells Clevers, (2015)&Slack (2018). Reproducing
of themselves, generating progeny designated to differentiate into
functional cell type, persisting for long time and behavior regulation by
the immediate environment.
• Stem cell culture are used in drug delivery or future therapeutic
applications .
• It provides properties for tissue repair, replacement and regeneration.
Stem cell transplants are used to replace the damaged cells and gene
some patients a new immune system.
• 3D cell culture and CRISPR gene editing techniques may advance in stem
cell research and treat disorders.
• Environmental pollutants, competition with agriculture and lack of fish
meal and oil for use in fish feeds the production of aquaculture will not be
sustainably in the future.
• These impacts can be avoided by using stem cell technologies such as;
surrogate broodstock, endangered fish protection and production, fish
meat production from stem cells, monosex fish production and gene
transfer studies.

• Embryonic stem cells are able to generate a mature
fish. After fertilization, cells start to divide by
mitosis and increase in number. As a result
generating an embryo at mid-blastula stage, only
one cell in this embryo has the power to generate
an adult fish

GERM STEM CELL TRANSPLANTATION IN AQUACULTURE
• In Salmonidae, Yoshizaki et al (2002) first developed
germ stem cell transplantation technology.
• Primordial germ cells (PGCs) are undifferentiated
stem cells that move into gonadal anlage
(gonocytes) during embryogenesis and after
reaching gonadal sides, they differentiate into a
mature egg in females or a mature sperm in males
(Çek, 2006; Çek et al., 2016).

Cont...
• Germ cell transplantation techniques used in fish. A) PGCs transplantation in embryos.
Donor derived PGCs are microinjected into embryo at mid-blastula stage. These embryos
must be grown until they are matured and produced donor-derived gametes. B) PGCs
transplantation into newly hatched larvae. At this stage, endogenous PGCs are still migrating
to the gonadal anlage. Immunolgical development has been developed and the larvae
cannot reject PGCs. C) Germ cell transplantation in adult fish, which have been previously
sterilized. This fish generate viable donor gametes. Modified from Lacerda et al., 2013; Çek
et al., 2016).

Cell-based fish meat production
• Singapore is the first country in the world, which allowed stem cell-based meat product for safe
consumption and commercial sale (Waltz, 2021). The stem cell-based chicken products developed by the
company Eat Just were available on the market in Singapore in 2020 (Ye et al., 2022). The USA is the
second country allowing stem cell-based products as safe consumption and sale. Wang et al., (2023),
• https://blogs.ifas.ufl.edu/fshndept/2022/04/11/alternative-protein-sustainable-cell-based-seafood/.

Companies Working on Cell-Based Seafood Production

• Early-life in vivo genome-wide CRISPR screening
for disease resistance by targeting GSCs and
subsequent transplantation using surrogate
technology in fish (Jin et al., 2021)

• Stem cell: Following fertilization the developing embryo contains
• a group of cells known as the inner cell mass that we eventually go
• on to form all tissues of the body however at this early stage of development
• these cells have not yet committed to become a specific
• type of cell is said to be in an undifferentiated state if the cells of the
• inner cell mass are isolated that they can grow infinitely in a plastic
• fish and maintain this undifferentiated state these cells are known
• as ES cells. Developing embryo ES cells retain the ability to form
• any type of cell in the adult body.

Stem Cell
Type
Description Examples
Totipotent Each cell has the capability to
develop into a new individual
Cell from early embryos
Pluripotent Cells can from any cell type Some cells of the
blastocyte
Multipotent Cells can distinguish, but from
different other tissues
Fetal tissue, adult
stem cell and cord
blood
*Medaka has given rise to the first embryonic stem (ES) cells besides the mouse, the first
adult testis-derived male stem cells spermatogonia capable of test-tube sperm
production, and most recently, even haploid ES cells capable of producing Holly, a semi-
cloned fertile female medaka from a mosaic oocyte created by microinjecting a haploid ES
cell nucleus directly into a normal oocyte.
*These breakthroughs make medaka a favoring vertebrate model for stem cell research

Protocol for es cell culture in fish
• Protocol for es cell culture in fish
• Collection of the fertilised eggs
• Blastula stage embryos are observed under a microscope and
embryos are immediately transferred to 35 mm small petri
dish.
• A batch of 25 to 30 embryos are first disinfected with 70%
ethanol and then wash five times with sterile phosphate buffer
saline in the Petri dish.
• The inner cell mass is harvested by tearing the Chiron with
fine forceps, and the chorion and eggshells are removed.
• Single cells are planted through gentle pipetting, and cells are
transferred to a new gelatine coated cell culture flask under
ES cell conditioned medium
• Maintenance of cells and characterization

Cont...
Subculture
• Take 2 ml of trypsin = EDTA, 5 ml of buffered saline solution,
4 ml of trypsin neutralizing solution.
• Rinse the flask with 5 ml of BSS and remove the BSS from
the flask
• Then 2ml of trypsin/EDTA solution is added to the individually
T25 flask. Permit the trypsinization to continue until
nearly 90% of the cells have been detached from the surface.
Hit the flask on the palm of the hand to detach the detached
cells. If only a few cells detach, wait 30 seconds and repeat
this
• After cells have been detached, neutralizes the trypsin in the
flask with 4 ml of trypsin neutralizing solution.
• Transfer the detached cells to a strike 15ml centrifuge tube
and rinse the flask with a final 2ml of BSS. Add this rinse to
the centrifuge tube.

Medaka Fish gills contain homeostatic and growth
stem cells (Ref: Julian Stolper et al. 2019)
• (A) Enucleated entire gills of medaka at different post-
embryonic times show that organ size increases during
post-embryonic growth (left). A gill contains four pairs of
branchial arches

Zebrafish employ cellular shield to protect blood stem cells from
damaging ultraviolet light
• Researchers observed that cells called melanocytes were
positioned above the stem cells in an umbrella pattern, and
subsequently showed this umbrella served as a shield
protecting stem cells from damaging ultraviolet (UV) light.
• When stem cells divide, they can form more stem cells or
other cells that perform specialized functions.
• The blood stem cell niche is crucial for regulating the
process of new blood cell formation, and its location varies
among species—for example, it is found in the bone
marrow of adult mammals and in the kidney of the
majority of fish species

Application of marine collagen for stem-cell-based therapy and
tissue regeneration
• Marine collagen and cartilage regeneration
• Cartilage defects are difficult to repair by themselves due to their
avascular nature.
• The transplantation of mesenchymal stem cells is a favorable
approach due to their high proliferative activity and their capacity to
differentiate into chondrocytes, which are responsible for cartilage
synthesis and maintenance.
• Natural, biodegradable scaffolds are mainly comprised of collagen,
silk, agarose, chitosan, hyaluronic acid, alginate and gelatin.
• Natural polymers, such as fibrin and collagen, confer high levels of
biocompatibility and can promote cell adhesion without incurring
immune responses.
• In particular, marine collagen has been widely used for chondrogenic
differentiation in the field of cartilage regeneration.

Marine collagen and bone regeneration
• Bone biomaterials that have been used as scaffolds for
bone tissue engineering include natural/synthetic
polymers, bioactive ceramics, decellularized extracellular
matrices, biodegradable metals or composites of any those
aforementioned.
• Among these, fish collagen has been attracting substantial
research interest as an adequate alternative to mammalian
collagen for bone tissue engineering.
• Tilapia is an important farmed freshwater species of fish
that has become one of the most popular sources of
collagen for bone tissue engineering.

Cont...
Tetracycline adsorption by tilapia fish bone-
based biochar: Mass transfer assessment and
fixed-bed data prediction
Physicochemical and bone regeneration
studies using scaffoldings of pure natural
hydroxyapatite or associated

Marine Drugs| Cosmetic, Biomedical and Pharmaceutical Applications
of Fish Gelatin/Hydrolysates

Marine Drugs: Cosmetic, Biomedical and Pharmaceutical Applications
of Fish Gelatin/Hydrolysates

Tilapia byproducts: commercial uses of the tilapia processing and
filleting industries’ waste (Ref: Tilapia Market)
• Biodiesel: It is made from vegetable oils and animal fats in order to
replace diesel. This biofuel is biodegradable and non-toxic, as well as, it
has lower emissions than the petroleum diesel. Researchers extracted
fish oil from different tilapia waste, such as viscera, fins, heads, skin,
scales and a mix of all the products. They evaluated the yield and acid
number of tilapia oil according to the used type of waste. Also, they
examined the potential of these extracted oils for biodiesel production.
According to their findings, the highest potential obtained from the
viscera that produced 217L of biodiesel per ton of processed waste.
The tilapia visceral oil is low in moisture and free fatty acids (FFA)
content, which is important for the feasibility of transesterification.
Tilapia viscera byproducts could be converted into a high-yield
biodiesel.
• Fish glue: This natural glue has been used by artists and craftsmen
since antiquity. It has been used in the gluing of different materials, like
wood, paper, cardboard and leather. It is a reversible glue for this
reason has been used mainly in creative projects of furniture and
woodwork. It is formulated from fish heads, bones and skins. In 2017, a
group of researches prepared very good quality glue from tilapia
wastes. In their research , the glue production is proposed as an
alternative way to enhance the value added of the fish for the new
product, to reduce the cost of the eliminated garbage and as a solution
to the environmental impact of the fish processing industries.
• Other products: The by-products generated from industrial filleting of
tilapia can produce other products such as surimi (minced fish) or
gluten free tapioca starch. Both products could be obtained from
mechanically recovered fish meat that otherwise would be discharged
into the environment.
• Medical biomaterials: Various pharmaceutical products and nutritional
supplements contain collagen. The primary structure of collagen is
formed by units of glycine-proline-hydroxyproline. The most common
sources of collagen are bovine and pig skins or chicken wastes.
However, animal-borne diseases urged the need of alternative collagen
sources, such as marine animals. Collagen can be extracted from fish
scales or skin. According to Qiang Huang, R., et al. (2011), every year in
China about 300 thousand tons of fish scales are discarded with the
total of 2 million tons of fish waste. These huge quantities of scales
could produce great amounts of collagen which could be further used
for medical, cosmetics and nutritional purposes. Additionally, tilapia
skin is a byproduct that contains about 27.8% collagen and can be used
for the extraction of collagen increasing the economic value of tilapia
processing industries. Tilapia skin collagen, due to its biological
properties, can be used to develop healing biomaterials, cosmetics or
food supplements. Already tilapia collagen powder from tilapia skin or
scales is available in the market. The price per kilogram is varied from
30 to 100€, according to the prices given by the Alibaba global trader.
Moreover, tilapia skin collagen sponge and electrospun nanofibers
were tested as wound dressing. In tilapia skin exist large quantities of
collagen proteins, types 1 and 3, which are important for scarring. The
collagen of tilapia skin is capable to heal wounds rapidly and
effectively. This treatment has been registered for an international
patent for treating burn wounds and already many kids and adults
have been benefited by this treatment.
• Leather: Many companies around the world are producing various
products from tilapia skin leather, such as shoes, wallets, vests, bags,
jewelry, belts and many other goods . In 2011, the trend of tilapia
leather products attracted the attention of the famous shoe designer,
Manolo Blahnik, who created a special line of shoes from fish skin; the
value per pair of shoes was 792€! The cost of tilapia leather products is
not as cheap as we could expect; the price of a wallet from tilapia skin
leather is around 90€, while its price as a texture varies from 1 to 3€
per piece (25-30cm length, 0.5-0.7mm/0.6-0.8mm/0.7-0.9mm
thickness).

Development of fish collagen in tissue regeneration and drug delivery
• Marine-based collagen as a
resealable replacement for
mammal-based collagen
Collagen distribution in the different parts of
the human body. Created with BioRender.com.
General preparation method for marine collagen. 1. Selection of fish
parts as collagen source; 2. Acid treatment or Pepsin treatment;
3. Purification steps; 4. Dialysis and freeze-drying; 5. Acid-soluble
collagen (ASC) and Pepsin soluble collagen (PSC). Created with
BioRender.com.
Different potential marine sources for collagen
synthesis. Created with BioRender.com.

Marine collagen and periodontal regeneration
• combination of biomaterials, growth factors and stem cells to
attain periodontal regeneration
• Biomaterials can provide templates to facilitate the
regeneration of periodontal tissues.
• Biomaterials, such as synthetic organic materials (examples
include poly-lactic-co-glycolic acid and poly-lactic acid) and
natural organic materials (examples include collagen, silk
fibroin, chitosan and alginate), have been extensively
investigated for the possibility of periodontal regeneration.
• Due to the reduced risk of zoonotic disease transmission and
the readily available reservoir of industrial fish waste from
which collagen can be extracted, fish collagen has attracted
attention over the years for use in the production of tissue-
engineered biomaterials for periodontal regeneration

Marine collagen and corneal regeneration
• In total, 10 million individuals worldwide are affected by corneal-related blindness.
• Full or partial corneal transplantation is typically the only effective treatment to
restore vision.
• Since the human corneal stroma is rich in type I collagen, collagen has been
postulated to be an ideal material for corneal scaffolds.
• One important source of collagen is fish scales, whereby scales of tilapia cultured
under controlled conditions mainly consist of type I collagen that align in a manner
identical to that observed in human corneas.
• As a result, this type of fish scale collagen may offer a viable and economical
substitute that can facilitate corneal regeneration.
• a scaffold with collagen was collected from the fish scales of Lates calcarifer for
corneal tissue engineering before its physiological, mechanical and cultural
characteristics in a scaffold were evaluated following co-culture with corneal cells.
• The cultured corneal cells were examined by reverse transcription-quantitative PCR
for putative stem cell markers.
• fish scale-derived collagen matrix was used to grow corneal epithelial and stromal
cells. No cytotoxicity was observed and primary human corneal epithelial cells
cultured on the fish scale-derived collagen matrix stained positive for cytokeratin
3/12 and the stem cell marker p63, suggesting that the correct phenotype of primary
human corneal epithelial cells was obtained following culture on this fish scale-
derived collagen matrices (FSCM).

• Other applications:
• Marine collagen and neural regeneration
• Marine collagen and oral mucosa regeneration
• Marine collagen and their immunomodulatory
properties in mesenchymal stem cells
• Marine collagen and skin regeneration
Implications for wound dressing
• Marine collagen in tissue regeneration

References
• https://www.google.com/search?q=fish+stem+cell&tbm=isch&ved=2ahUKEwjqpdfI4vCEAxXaxzgGHVxLBTYQ2-
cCegQIABAA&oq=fish+stem+cell&gs_lp=EgNpbWciDmZpc2ggc3RlbSBjZWxsMgcQABiABBgYSKUlUI0VWPAZcAB4
AJABAJgBbqAB9gOqAQMzLjK4AQPIAQD4AQGKAgtnd3Mtd2l6LWltZ8ICBBAjGCfCAgUQABiABMICBhAAGAcYHsIC
CRAAGAcYHhjHA8ICCBAAGAUYBxgewgIIEAAYCBgHGB6IBgE&sclient=img&ei=_FzxZeq3BtqP4-
EP3JaVsAM&bih=739&biw=1600&prmd=ivsnbmtz#imgrc=MgwbUuTPOtX2RM
EP3JaVsAM&bih=739&biw=1600&prmd=ivsnbmtz#imgrc=Q7Qy8GJpWAJKLM
EP3JaVsAM&bih=739&biw=1600&prmd=ivsnbmtz#imgrc=QqIbxIMiE8_IiM
• Şehriban ÇEK YALNIZ and Fatmagün AYDIN. 2023. FISH STEM CELL TECHNOLOGY IN AQUACULTURE . h
ttps://www.researchgate.net/publication/374977673 .
• https://tilapiamarket.rodaint.com/tilapia-byproducts-commercial-uses-of-the-tilapia-processing-and-filleting-
industries-waste/
• https://www.newscientist.com/article/2373850-zebrafish-produce-sunscreen-to-protect-their-embryos-from-
uv-light/
• Jelous Monika E. 2022. Fish Stem Cell Culture Acta Scientific Veterinary Sciences (ISSN: 2582-3183)
• https://www.frontiersin.org/articles/10.3389/fcvm.2019.00107/full
• https://doi.org/10.7554/eLife.43747.002
• https://www.niddk.nih.gov/news/archive/2018/zebrafish-employ-cellular-shield-protect-blood-stem-cell-uv-
light#:~:text=While%20studying%20the%20blood%20stem,damaging%20ultraviolet%20(UV)%20light.

Thank you for your time B
Zebrafish as a Smart Model to Understand Regeneration After Heart
Injury: How Fish Could Help Humans (Ref: Giorgia Beffagna 2029)

Fish stem cells and their applications by B

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