Stem

cells
Presented By:
Nehal gamal
◆
Contents :
Definitions of stem cells
◆ Characteristic features of stem cells
◆ Types of stem cells
◆ Dental stem cells
◆ Isolation of stem cells

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 Definitions of stem

cells
A stem cell is the a cell with the
unique ability to continuously divided
:

and differentiate (develop) into

various other kind(s) of cell & tissues
aiding as a repair system for the body.
 Found in all multicellular organisms.

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 Definitions of stem
 cells
When a stem cell divides, each cell
has the potential to either remain a
stem cell or become another type of
cell with a more specialized
function (i.e. a muscle cell, a red
blood cell, a brain cell …etc.)

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 telomeres
 Telomeres are distinctive structures
found at the ends of our
chromosomes. They consist of the
same short DNA sequence repeated
over and over again.

 They consist of the same sequence of

bases repeated over and over.

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 In humans the telomere sequence is TTAGGG.
 This sequence is usually repeated about 3,000 up to
15,000 times base pairs in length.

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 Functions of
telomeres
1. They protect the ends of our
chromosomes by forming a cap,
much like the plastic tip on
shoelaces. If the telomeres were not
there, our chromosomes may end up
sticking to other chromosomes.

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Telomeres appear
as the bright
spots at the ends
of each
chromosome in
the picture shown
above

Image credit: "Telomere caps," by U.S. Department of Energy Human Genome Program (public domain)
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2-Every time a cell carries out DNA replication the chromosomes are
shortened by about 25-200 bases (A, C, G, or T) per replication.

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 Functions of
telomeres
3- DNA is left undamaged,because their ends are
protected by telomeres, the only part of the
chromosome that is lost, is the telomere.

4-Without telomeres, important DNA would be lost

every time a cell divides (usually about 50 to 70
times).

5-This would eventually lead to the loss of entire

genes.

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 Mechanism of action of
telomere :
 Telomeres need to be protected from a cell's
DNA repair systems because they have single-stranded
overhangs, which "look like" damaged DNA.
 The overhang at the lagging strand end of the chromosome
is due to incomplete end replication The overhang is actually
generated by enzymes that cut away part of the DNA.

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• In some species (including humans),
the single-stranded overhangs bind to
complementary repeats in the nearby
double-stranded DNA, causing the
telomere ends to form protective
loops^33cubed.
• Proteins associated with the telomere
ends also help protect them and
prevent them from triggering DNA
repair pathways

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 The repeats that make up a telomere are eaten away
slowly over many division cycles, providing a buffer that
protects the internal chromosome regions bearing the
genes (at least, for some period of time).
 Telomere shortening has been connected to the aging of
cells, and the progressive loss of telomeres may explain
why cells can only divide a certain number of times.

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 Telomerase
• Some cells have the ability to reverse telomere
shortening by expressing telomerase, an enzyme that
extends the telomeres of chromosomes.

• Telomerase is an RNA-dependent DNA polymerase,

meaning an enzyme that can make DNA using RNA as
a template

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 Mechanism of telomerase:
1-The enzyme binds to a special
RNA molecule that contains a
sequence complementary to the
telomeric repeat.
2-It extends (adds nucleotides to) the
overhanging strand of the telomere
DNA using this complementary
RNA
as a template.

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Mechanism of telomerase:
3-When the overhang is long enough, a matching strand can
be made by the normal DNA replication machinery (that is,
using an RNA primer and DNA polymerase), producing
double-stranded DNA.
4-The primer may not be positioned right at the chromosome
end and cannot be replaced with DNA, so an overhang will
still be present. However, the overall length of the telomere
will be greater.

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 Telomerase
• Telomerase is not usually active in most somatic cells (cells of the body),
but it’s active in germ cells (the cells that make sperm and eggs) and
some adult stem cells. These are cell types that need to
undergo many divisions, or, in the case of germ cells, give rise to a new
organism with its telomeric “clock” reset.

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 Telomerase
• Interestingly, many cancer cells have shortened
telomeres, and telomerase is active in these cells. If
telomerase could be inhibited by drugs as part of
cancer therapy, their excess division (and thus, the
growth of the cancerous tumor) could potentially be
stopped.

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 Characteristics of
◆ Stem cells are distinguished from other cell types by
stem
specific cell
important Characteristics:

1-Self –renewal: they are

unspecialized cells capable of
. renewing themselves through cell
division

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 Characteristics of
stem cell
2-Unlimited-potency.
 It is the capacity to differentiate into specialized cell
types.
 Under certain physiologic or experimental conditions,
they can be induced to become tissue or organ specific
cells with special functions.
3-Unspecialization.

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 Characteristics of
stem
4-Homingcell
 It refers to the ability of circulating stem cells or
exogenously administered stem cells to locate and enter an
environmental niche.

 Throughout its life span, a stem cell may migrate between

niches during embryonic development and also during the
adult life span

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Characteristics of
stem cell
5-Engraftment:
 These cells you received on transplant day know where
they belong in your body.

 They move through your bloodstream into your bone

marrow. When these cells begin to grow and make new
blood cells.

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Homing

Engrafting

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 Classification of Stem cell according to
their potency
• Totipotent stem cells
1. They are produced by the first few divisions of the
fertilized egg. Early (1-3 days) embryo is totipotent.

2. These cells can differentiate into embryonic and extra

embryonic cell types and placenta.

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Classification of Stem cell according to
their potency
 Pluripotent stem cells (PSCs) are the descendants of
totipotent cells and can differentiate into cells derived
from the 3 germ layers

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Sources of pluripotent human
embryonic stem cells:
 They are derived from the inner mass of early human
blastocysts.
 Primitive germ cells with the characteristics of pluripotent
cells are isolated from developing embryos.

 Reprogramming factors can induce the differentiation of

adult somatic cells (e.g., skin fibroblasts) into pluripotent
stem cells.

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• Multipotent stem cells can produce only cells of a closely
related family of cells (e.g. hematopoietic stem cells
differentiate into red blood cells, white blood cells.
• Unipotent stem cells can produce only one cell
type, but have the property of self-renewal which
distinguishes them from non-stem cells .
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 types of stem cells according to
source of origin:
There are three main types of stem cells
1.Embryonic stem cells(ESCs)
2.Adult stem cells(tissue stem cells)
3.Induced pluripotent stem cells(iPSCs)

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 Embryonic
◆ A blastocyst (BLAST-oh-sist
stem ), is a
cells(ESCs)
totipotent, pre-implantation embryo that
develops 5 days after the fertilization of an
egg by a sperm.
◆ It contains all the material necessary for the
development of a complete human being.
◆ The blastocyst is a mostly hollow sphere of cells
that is smaller than the period at the end of this
sentence.

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 Embryonic stem
cells
 In its interior is the inner cell mass,
which is composed of 30-34 cells that
are referred to by scientists as
pluripotent because they can
differentiate into all of the cell types
of the body.

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 Embryonic stem
 Although theycells
have the greatest biological
potential, ethical issues on the use of ESCs
has precluded their widespread study,
especially in humans
 Sources of Embryonic
stem cells
In vitro fertilization Nuclear transfer

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◆
In vitro
The largest potential source of blastocysts for stem cell research is from in vitro

fertilization :
fertilization (IVF) clinics, The process of IVF requires the retrieval of a woman’s eggs
via a surgical procedure after undergoing an intensive regimen of “fertility drugs,”
which stimulate her ovaries to produce multiple mature eggs
◆ Because not all the fertilized eggs are implanted, this has resulted in a large bank of
“excess” blastocysts that are currently stored in freezers around the country
 The blastocysts stored in IVF clinics could prove to be a major source of embryonic stem
cells for use in medical research.
 However, because most of these blastocysts were created before the advent of stem cell
research, most donors were not asked for their permission to use these left-over
blastocysts for research.

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In vitro
fertilizati
on:
 Nuclear transfer
 In animals, nuclear transfer has been accomplished by inserting the
nucleus of an already differentiated adult cell
 for example, a skin cell—into a donated egg that has had its nucleus
removed. This egg, which now contains the genetic material of the
skin cell, is then stimulated to form a blastocyst from which
embryonic stem cells can be derived.
 The stem cells that are created in this way are therefore copies or
“clones” of the original adult cell because their nuclear DNA
matches that of the adult cell.
 ar
transf
er
 Adult stem cells
 Also called tissue-specific or somatic stem cells.

 They are derived from postnatal fully developed tissue

 Compared with ESCs, adult derived stem cells have several

limitations with respect to lifespan and differentiation
potential .
Sources of Adult
stem cells
Stem cells are present inside different types of tissue
including:
 the brain
 bone marrow
 blood and blood vessels
 skeletal muscles
 skin
 the liver

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 Induced pluripotent stem
cells(iPSCs)
 These are reprogrammed somatic cells having
pluripotency like ESCs.

 This might provide an alternative pathway which

eliminate the ethical issues regarding the use of tissue
from human embryos and allow to overcome problems
of rejection after non-autologous cell implantation.
 Induced pluripotent stem
cells(iPSCs)
 They were expected to become the important tool in the
advancement of personalized medicine.

 Basically, iPSCs have been generated by reprograming cells

via incorporation of several genes and they have similarities
to human ESCs in their morphologies, gene expression, in-
vitro differentiation potential and teratoma formation

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 Sources of Induced pluripotent
stem cells(iPSCs)
Animals
source

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Dental stem cells

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 Dental stem cells
 Oral tissues have been considered a potential source of multipotent stem
cells like populations. With the exception of enamel, which lacks
ameloblasts or other cellular elements following tooth development.
 The periodontium and dentine continue to retain some
regenerative or reparative capacities.

 Dental multipotent stem cells are indicated for the

regeneration of the dentin-pulp complex, bone, cartilage
and neuronal tissues

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 Different populations have been isolated and characterized in
postnatal dental tissues and classified according to the tissue of origin
into:
1- Dental pulp stem cells (DPSCs)
2- Stem cells from exfoliated deciduous teeth (SCED).
3- The cells located in the apical papilla (root foramen area)
4-The periodontal ligament.
5- Dental follicle.
6-Gingiva.
7-Oral epithelial stem cells are derived from basal layer of the
epithelial oral mucosa.
 1- Dental pulp stem cells

(DPSCs)
They are ectomesenchymal multipotent stem cells.
 They are easily available from discarded teeth after extraction.
 The differentiation potential of DPSCs from natal teeth to
1. Adipogenic.
2. Osteogenic.
3. Chondrogenic.
4. myogenic .
5. neural-glial cell lines
6. osteoblasts .
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1- Dental pulp stem cells
(DPSCs)
 Sometimes DPSCs are referred to as odontoblastoid cells, because
they appear to synthesize and secrete dentin matrix like the
odontoblasts that they replace.
 When transplanted in vivo the cells derived from dental pulp
generated functional dental tissues similar to those generated by
the cells of the dentin/pulp complexes.
 When cultured in ceramic substrates, such as hydroxyapatite or
tricalcium phosphate; the cells are able to form bone, dentin and
cementum like tissues .
2- Stem cells from exfoliated deciduous
teeth (SCED).
 They are a heterogeneous multicellular
population of stem cells identified as
highly proliferative clonogenic cells
capable of differentiating into a variety of
cell types including
 neural cells.
 Adipocytes.
 odontoblasts.
2- Stem cells from exfoliated
deciduous teeth (SCED).
 Immature dental pulp stem cells can be extracted
from the pulp of primary teeth.
 These cells can differentiation into
1. smooth and skeletal muscles,
2. neurons.
3. Cartilage.
4. bone under chemically defined culture conditions .

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3- The cells located in the apical papilla
(root foramen area)
 These cells show an ability to differentiate into
cells of the osteogenic
 odontogenic.
 adipogenic.
 neurogenic lineages.
3- The cells located in the apical papilla
(root foramen area)
 It is thought that stem cells from apical papilla.
 May be responsible for the formation of primary
odontoblasts that account for the formation of root
dentin.
 They also showed other favorable characteristics, such
as telomerase(cellular immortality) and improved
migration capacity .

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 Protocol of isolation of apical
foramen stem cells
1. Normal human impacted third molars
were collected from the Dental Clinic.

2. the apical papilla was minced and

digested in a solution of 3 mg/mL
collagenase type I (Invitrogen, Carlsbad,
CA) and 4 mg/mL dispase (Invitrogen) for
40 minutes at 37°C.

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Protocol of isolation of apical
foramen stem cells
3-Single-cell suspensions were obtained
by passing the solution through a 70-µm
cell strainer.

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4- Cells were seeded at 1 × 104 cells/ well into 6-well plates .

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5- then cells were cultured with a standard medium defined as α-
MEM (Invitrogen) supplemented with 15% fetal bovine serum
(FBS) (Hyclone, Kerrville, TX), 100 mol/L L-ascorbic acid 2-
phosphate, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100
µg/mL streptomycin at 37°C in 5% CO2.

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6- The medium was changed every 2–3 days.
7-After 7–10 days, the attached cells were collected and
prepared for limiting dilution procedures to obtain single-
colony-derived strains

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7-The cell suspensions were diluted such that each well of the
96-well plate was seeded with approximately 1 cell.
8-A number of these single-colony-derived strains were pooled,
cultured, and passaged at 1:3 ratio when they reached 80%
confluence. The experiments were carried out by using cells at
passages 3–12.

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9-For the induction of adipogenesis, cells were exposed to
adipogenic medium (α-MEM supplemented with 1 µg/mL
insulin, 1 µmol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-
methyl-xanthine, and 10% FBS) for 3 weeks.

10-The oil droplets that accumulated inside the cells were

visualized by staining with oil red O reagent

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Nada OA, El Backly RM. Stem Cells From the Apical Papilla (SCAP) as a Tool for
Endogenous Tissue Regeneration. Front Bioeng Biotechnol. 2018 Jul 24;6:103. doi:
10.3389/fbioe.2018.00103. PMID: 30087893; PMCID: PMC6066565
Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human
dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S
A. 2000 Dec 5;97(25):13625-30. doi: 10.1073/pnas.240309797. PMID:
11087820; PMCID: PMC17626
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 4-The periodontal ligament stem
cells (PDLSCs).
 They are capable of differentiating into :
1. cells resembling cementoblasts,
2. osteoblasts,
3. adipocytes,
4. Chondrocytes.
5. fibroblasts.

 This population has a faster cell growth rate and higher

clonogenic capability than bone marrow MSCs.
 4-The periodontal ligament stem
cells (PDLSCs).
 Periosteum derived stem cells lie in the thinner membrane
of the periosteum.
 They can differentiate into
1. Osteoblasts.
2. adipocytes.
3. chondrocytes.
 But, they have limited potential for cell differentiation

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 5- Dental follicle.
 Dental follicle is a loose connective tissue sac surrounding
the enamel organ and the dental papilla of the developing
tooth germ before eruption.

 The stem cells were first isolated from the follicle of

human impacted third molars.

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 Dental follicle
 In vitro, dental follicle precursor cells They can
differentiate into
1. Osteogenic.
2. odontogenic.
3. Cementogenic.

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 6-Gingiva.
 Gingiva represent a source of stem cell.
Progenitor cells and multipotent stem
cells subpopulations have been isolated
from gingival fibroblasts.

 These fibroblasts are easily accessible

and have used to induce pluripotent
stem cell lines .
 Adult human cells are
reprogrammed to form embryonic
stem like cells called induced
pluripotent stem cells (IPSCs).

 Oral fibroblasts are able to form

IPSCs in lab applicable for future
use

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 7-Oral epithelial stem cells
 They are derived from basal layer of the epithelial oral mucosa.
 They are unipotent stem cells and possess clonogenicity. They
can form highly stratified and well organized graft .
7-Oral epithelial stem cells
 Salivary gland stem cells derived from the stromal tissue of
salivary glands. They are useful for regeneration of salivary
gland damaged from irradiation and can be guided to
1. osteogenic,
2. chondrogenic
3. adipogenic differentiation.

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1. Prepare the Enzyme Solution and Proliferation
Medium (PM)
1. Make Collagenase Type I Solution: Weigh out collagenase type I
(12 mg/ml) and dissolve in 1 ml PBS and filter using a 0.2 μm
syringe filter. Then place it 15 ml tube and keep it at -20 °C until
needed.

2. Make dispase Solution: Weigh out dispase (16 mg/ml) and dissolve
in 1 ml PBS and filter using a 0.2 μm syringe filter. Then place it 15
ml tube and keep it at 4 °C until needed.

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1. Prepare the Enzyme Solution and Proliferation
Medium (PM)
3. Make enzyme solution: Add 1 ml collagenase type I solutions (12
mg/ml) and 1 ml dispase solutions (16 mg/ml) into the 2 ml sterile PBS
containing 100 mg/ml penicillin, 100 mg/ml streptomycin. Total
concentration of dispase and Collagenase I in final volume should be 4
mg/ml and 3 mg/ml, receptively. Then, aliquot this volume in to four
15 ml tube, each containing 1 ml enzyme solution. Each tube could be
used for one pulp digestion.

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1. Prepare the Enzyme Solution and Proliferation Medium
(PM)
4. Make washing solution: Add 100 mg/ml penicillin, 100 mg/ml
streptomycin into PBS.
5. Make basic media: Alpha modification of Eagle's medium (α-MEM)
supplemented with 10% FBS & 100 units/ml penicillin, 100 mg/ml
streptomycin.
6. Make the Proliferation Media (PM): Alpha modification of Eagle's
medium (α-MEM) supplemented with 10% FBS, 100 μM L-ascorbic
acid 2-phosphate, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml
streptomycin, 0.25 mg/ml amphotericin B.

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1. Prepare the Enzyme Solution and
Proliferation Medium (PM)
7. Make Odontogenic media: Alpha modification of Eagle's
medium (α-MEM) supplemented with 10% FBS, 100 μM L-
ascorbic acid 2- phosphate, 2 mM L-glutamine, 100 units/ml
penicillin, 100 mg/ml streptomycin, 0.01 μM Dexamethasone, 5 mM
β-Glycerol phosphate, 1.8 mM Monopotassium phosphate

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Pilbauerová N, Soukup T, Suchánková Kleplová T, Suchánek J.
Enzymatic Isolation, Amplification and Characterization of Dental
Pulp Stem Cells. Folia Biol (Praha). 2019;65(3):124-133.

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 2. Prepare Human Dental Pulp Tissue
for Dental Pulp Stem Cell Isolation
1. Normal human third molars were collected from adults (21-29 years of
age) from the Dental Clinic
2. Teeth were placed into the basic medium (α-MEM supplemented with
10% FBS) .
3-They were transferred into laboratory at 4 °C. Under the sterile
condition, working within a biohazard laminar flow hood, set-up was
done one 100 mm Petri dishes for each tooth to be processed.
4-Tooth surfaces were cleaned by 70% ethanol.

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 2. Prepare Human Dental Pulp
Tissue
5. While working in one of the Petri-dishes, tooth was kept by dental
forceps and a scalpel blade was used to clean off debris and
periodontal ligament on the roots.
6. Cemento-enamel junction was slowly cut by using sterilized dental
disc to reveal the pulp chamber. It should be considered that cutting
process must be performed slowly to reduce overheating of the dental
tissue.
7. Pulp tissue was gently separated from the crown and root.
8. Pulp tissue was cut into small pieces with the aid of a scalpel blade

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 3. Human Dental Pulp Isolation
 The methods most frequently used for DPSCs isolation
are
1. Enzymatic dissociation of pulp tissue
2. Direct cell outgrowth from pulp tissue explants

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Enzyme-digestion method
1. Small pieces of pulp tissues were transferred
into Enzyme solution for 1 hr at 37 °C. Vortex every
30 min to help breaking up tissue.
2. Afterwards, large cell aggregates were removed
and Single-cell suspensions were obtained by
passing cells through a 70-μM cell
strainer.
3. Single-cell suspensions were centrifuged at
1,200 rpm for 5 min at room temperature.
4. Supernatants were carefully pipetted off and
pellet was re-suspended in 1 ml proliferation
medium (PM). (Note: FBS in proliferation
medium terminate enzymatic dissociation.)
5. Single-cell suspensions of dental pulp was
seeded into 25 cm2
culture flask with PM and then incubated at 37 °C
in 5% CO2.
6. The culture medium was changed every three
days until the cell confluency was achieved.
explant outgrowth method
1. Pulp tissue was minced into 1-2-
mm fragments, and each piece was
placed in 25-cm2culture flasks with
PM
and then incubated at 37°C in 5% CO2
it should be considered that total
volume of the PM for outgrowth of
cell must support the attachment of
pulp pieces for
further cell outgrowth (2-3 ml per
flask).
2. Medium was changed after
outgrowth was observed.
3. The outgrown cells at confluence
were sub-cultured at ratio of 1:4
(passage 1).
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 Characterization of stem cells
 After isolation of stem cells, differentiating occurs through
various markers and by various methods.
 Majority of the studies used:
1. Phase contrast inverted microscope.
2. Flow cytometry .
3. RT-PCR.
4. Colony forming units.
5. Protein extraction and immunoblotting.

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Phase contrast inverted microscope
• Can examine live cells
• Can observe cells in Petri dishes or cell cultures
• Used in observing stem cell differentiation culture after cell-
seeding for general histology using GFP fluorescent proteins

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Flow Cytometry
• Measure cell properties as they flow in fluid suspension
across illuminated path
• Can be done by labelling Stem cell markers of interest
such as CD73 and CD146 which are associated with cell
growth with fluorescent marker
• Allows for quantitative and qualitative analysis of cell
populations

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 RT-PCR
• Reverse transcription-polymerase chain reaction is a
technique used for mRNA detection and quantitation.

• Converts RNA molecules into their complementary DNA

(cDNA) sequences‫ز‬

• Can be used to amplify gene expression from markers of

interest to identify DPSCs

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Colony Forming Units
• CFU assays allow measurement of the
proliferation and differentiation ability of
individual cells within a sample.
• Measures by the observation of the colonies
(consisting of more differentiated cells) produced
by each input progenitor cell.

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 CFU result Example
• Images are a representation of the following
CFUs
• colony forming unit-erythrocyte (cfu-E),
• blast-forming unit-erythrocyte ( bfu-E)
• colony-forming unit granulocyte
macrophage (cfu-GM) (neutrophils)
• colony-forming unit granulocyte
macrophage (cfu-GM)

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Western Blotting
• Western blotting is a technique that rely on binding
between protein and probe, usually an antibody, to
allow detection of the protein
• The bound protein-probe is run through
electrophoresis to allow for the analysis of the
protein

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Type of cells Source of isolation characterized marker
osteoblasts dental pulp, periodontal ligament, the common marker was
alveolar bone, apical papilla and STRO-1.
maxillary sinus OCT-4, NANOG and SOX-2

adipocytes dental pulp, PDL granulation STRO-1, ALP, CD29, CD34,

tissue, alveolar bone and apical CD44.
papilla

Odontoblasts differentiated from the various Similar to osteoblasts

tissues next to osteoblasts and markers
adipocytes.
neuroblasts dental pulp Nestin and Nucleostemin .
SOX-2
Fibroblasts maxillary sinus tissue STRO-1
Chondroblasts and dental pulp, gingival tissue and for both was
chondrocytes alveolar bone but later from PDL and STRO-1
PDL granulation tissue
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Preservation
 Once the tissue samples are obtained, they are
transferred under proper conditions to a tooth bank
where they are stored.
 The approaches used for stem cell storage are
Cryopreservation and Magnetic freezing
The approaches used for stem cell storage:
cryopreservation magnetic freezing technology

Def. is the process of preserving organelles, cells is referred to as cells alive system (CAS)
or whole tissues by cooling them at very
low temp.(typically -80°C using solid CO2
or -196°C using liquid nitrogen

principle 1- bring tissue cell to a zero metabolism and applying a weak magnetic field to water or
non-dividing state by reducing the temp. in cell tissue, which will lower the freezing point
presence of cryoprotectant (anti freeze). of that body by up to 6–7°C
It can be done :
Over solidCO2 at -79 °C .
Low temp. freezer at -80°C .
In vapor phase nitrogen at -150°C .
In liquid nitrogen at -196°C .

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 cryopreservation

Mechanism

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105
 1-Selection of plant material:
 Selection of plant material is important.

 Any tissue can be selected for this purposes.

 Biopsy samples of limited size (c. 1–3mm3 , up to 1 mm in

thickness) are likely to be suitable for immediate
cryopreservation.

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 1-Selection of plant material:
 Samples of ovarian or testicular tissue destined for subsequent
autologous transplantation can be stabilized by cryopreservation
and then stored until suitable arrangements for the
transplantation can be made.
 Larger tissue pieces may require further dissection or a level of
enzymic digestion prior to preservation .

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2- addition of cryoprotectant
 They are chemical which prevent
cryodestruction.
 These are sucrose, alcohols, glycols,
some amino
acid(proline),DMSO(dimethyl
sulfoxide).
 Generally two cryoprotectants should
be used together instead of single one
as they are more effective.

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3- freezing
 The sensitivity of cells to low temperature depends on the plant
species.
 There are four different types of methods:
1. Slow freezing method.
2. rapid freezing method.
3. combine freezing method.
4. Dry freezing method

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Slow freezing method
the tissue or plant material is slowly
frozen at slow cooling rate ,the
advantage is the plant cells are partially
dehydrated and survive better.

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rapid freezing method
It involves plunging the vials in
liquid nitrogen. The temperature
decreases from -300 to -1000 degree
rapidly.

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3-combine freezing method
this is combination of both slow and rapid methods.
The process is carried out in step wise like manner.

4-Dry freezing method in this method dehydrated cells and

seeds are stored

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4-storage in liquid nitrogen
 The maintenance of the frozen cells or material at specific
temperature is very important.
 In general the temperature is kept -70 to-196 degree.
 Prolong storage is done at temperature of -196 degree in
liquid nitrogen.
 To prevent damage, continuous supply of nitrogen is done.

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5- thawing
 Usually carried out by plunging the vials
into warm water bath with vigorous
swirling.
 As thawing occurs the vials are transferred
to another bath at 0 degree.

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6-washing and reculturing
 The preserved material is washed few times
to remove the cryoprotectant.

 This material is then recultured in afresh

medium.

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7- Measurement of viability

 Thereis possibility of death of cells due to

storage stress.
 Thus viability can be found at any stage .
 It is calculated by formula :
 n

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8- plant regeneration
 The viable seeds are cultured on non-
specific growth medium.
 Suitable environmental conditions are
maintained

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Thanks!

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