edited by : Qussai Abbas

1. Presented by:
Supervised by:

2. ?

4. I. Human ES cells can be maintained for months to years in
culture as undifferentiated cells {telomerase activity}.
II. Ten million or more human ES cells can be easily grown
and sampled at any time-point during differentiation into
specific cellular lineages {teratoma}.
III. Marker: SOX2, NANOG, Oct-4, SSEA-1, SSEA-3, SSEA-4
TRA-1-60, TRA-1-81 Frizzled5, CD90.
IV.Surface adherence.

5. • Derived from the inner cell mass (ICM) of the preimplantation blastocyst
• (day 5-7 of development ,150-200 cells).
• They give rise to the 3 germ layers: ectoderm, mesoderm and endoderm

6. Somatic cell
nuclear
transfer
(SCNT)
Potential Alternatives to ES cells

7. Nuclear
Reprogramming
Induced pluripotent
stem cells
(iPS)
Potential Alternatives to ES cells

8. Nuclear
Reprogramming
Induced pluripotent
stem cells
(iPS)
 Cellular reprogramming can be achieved by ectopic
delivery in somatic cells of four transcription factors
known as OSKM (OCT3/4, SOX2, KLF4, and c-MYC, with
or without Lin28) under specific culturing conditions.
 Takes 3-4 weeks to convert differentiated cell to
pluripotent stem cells.
Potential Alternatives to ES cells

9. Nuclear
Reprogramming
Induced pluripotent
stem cells
(iPS)
Potential Alternatives to ES cells

10. Nuclear
Reprogramming
Induced pluripotent
stem cells
(iPS)
Potential Alternatives to ES cells

11. CULTURING HUMAN EMBRYONIC STEM CELLS (ESCS)

12. 1. Sacrifie female mice using CO2 gas.
2. Open the peritoneal cavity by making a Y-incision and
dissect out embryos from the uterus.
3. Carefully transfer dissected embryos into petri dishes
containing sterile PBS.
4. Remove the head and internal organs from the
embryos carefully with sterile forceps and scissors.
5. Place all dissected embryos in a fresh dish of PBS and
cut tissue into approximately 1 mm pieces with the
scalpel.
MEFs used in human ES cell propagation are isolated
from E12.5–14.5 mouse fetuses.

13. 1. Sacrifie female mice using CO2 gas.
2. Open the peritoneal cavity by making a Y-incision and
dissect out embryos from the uterus.
3. Carefully transfer dissected embryos into petri dishes
containing sterile PBS.
4. Remove the head and internal organs from the
embryos carefully with sterile forceps and scissors.
5. Place all dissected embryos in a fresh dish of PBS and
cut tissue into approximately 1 mm pieces with the
scalpel.
MEFs used in human ES cell propagation are isolated
from E12.5–14.5 mouse fetuses.

14. 1. Sacrifie female mice using CO2 gas.
2. Open the peritoneal cavity by making a Y-incision and
dissect out embryos from the uterus.
3. Carefully transfer dissected embryos into petri dishes
containing sterile PBS.
4. Remove the head and internal organs from the
embryos carefully with sterile forceps and scissors.
5. Place all dissected embryos in a fresh dish of PBS and
cut tissue into approximately 1 mm pieces with the
scalpel.
MEFs used in human ES cell propagation are isolated
from E12.5–14.5 mouse fetuses.

15. 6. Add trypsin/EDTA solution and DNase if solution appears viscous .
7. After 40 min add mEF medium .
8. Centrifuge , Decant supernatant and resuspend the pellet using fresh
mEF medium.
9. Observe cells with the inverted microscope to make sure that they look
normal, and not apoptotic .

16. NOTE:
• To prevent mEFs from dividing when stem
cells are plated on them, they must be
inactivated with either irradiation (for
example cesium exposure) or mitomycin C .

17. 1. Obtain a frozen vial of hESCs from liquid-
nitrogen storage.
2. Thaw the vial in 37°C water bath for no more
than 1.5 min or until a small crystal is left.
3. Add hESCs to conical tube then centrifuge the
tube at 200 × g for 3 min at room temperature.
4. Decant the supernatant and gently break the
pellet using 1 mL of hESC medium.
5. Add the 1 mL of hESC suspension dropwise into
one well of a six-well plate or one T25-cm2 flask.
6. Replace mEF medium with hESC medium.

18. 1. Dulbecco’s modified Eagle’s medium (DMEM)
2. Non Essential Amino Acid (NEAA) solution
3. L-Glutamine/β-mercaptoethanol solution(β-ME) solution
4. Basic fibroblast growth factor (bFGF)
hES cells media:
ES cells were fed daily with fresh medium and were passed
onto fresh feeder plates.

19. hES cells on MEF
Immunofluorescence of
OCT4+ cells

20. hES cells on MEF
(C) Alkaline phosphatase.
(B) SSEA-4.
(A) SSEA-1.

21. 22
YOLK SAC

22. 23
Liver
&
Spleen

23. 24
Bone
marrow

25. S17 cells are stromal cell line derived from murine BM.
 Irradiated S17 cells are plated onto 0.1% gelatin-coated six-well plates .
 Feeder layers should be prepared at least 1 day prior to coculture with ES
cells and remain suitable for use up to 2 wk when kept in a 37°C, 5% CO2
incubator.

26. Coculture of hES Cells on S17 :
ES/S17 differentiation media:
1. DMEM.
2. FBS.
3. Non Essential Amino Acid (NEAA) solution.
4. L-Glutamine/β-mercaptoethanol solution(β-ME) solution.
5. Pencillin-Streptomycin (P/S).
 To improve the viability of human ES cells for hES cells/S17 cocultures,
small colonies or clusters of ES cells should be plated onto the S17 cell
rather than a single cell-suspension. To maintain small colonies rather
than single cells, collagenase type IV is used to harvest ES cells.

27.  place the plate in a 37°C, 5% CO2 incubator.
 Change the culture medium every 2–3 d.
 For the first few days, colonies typically maintain appearance of undifferentiated
hES cells.
 They then show obvious evidence of differentiation, as evidenced by three-
dimensional cystic structures and other loosely adherent structures.
Colonies of undifferentiated hES cells Three-dimensional cystic structures

28. • Embryoid bodies (EBs) are three-dimensional aggregates of
pluripotent stem cells.
• EBs are formed by the homophilic binding of the Ca2+
dependent adhesion molecule E-cadherin, which is highly
expressed on undifferentiated ESCs.
• EB formation is an important methodology when researchers
want to avoid
• more complex interactions with stromal cells

30. 02
Colony forming
cell assay
01Flow Cytometry
04
In vivo
engraftment
03RT-PCR

31. Harvest of Differentiated hES Cells From hES/S17
Cell Cocultures
TIME:
• Optimal time required for differentiation of human ES
cells into CD34+ cells varies depending on the hES cell
line and stromal cells used.
• On average, culture for 14–21 d produces the best
results for these purposes.
• A time-course experiment to sample cells every 2–3 d
is recommended to find the optimal time-point for
specific cells formed.

32. NOTE:
• For flow cytometry and colony-forming
assays, it is necessary to produce a single-
cell suspension of ES cells that have
differentiated on S17 or other stromal
cells.
Harvest of Differentiated hES Cells From hES/S17
Cell Cocultures
{collagenase ,trypsin–EDTA, filter, Trypan blue}

33. Flow Cytometric Analysis
FACS wash media:
PBS containing 2% FBS 0.1% sodium azide and Propidium iodide.
Protocols:
1. Wash cells one or twice with FACS media before starting staining.
2. Stain with either antigen-specific antibodies or isotype control on
ice.
3. If the first antibodies are unconjugated, the cells should be
incubated with conjugated secondary antibodies after washing
with FACS media between staining steps.

35. Hematopoietic Colony-Forming Cell Assays (CFCs)
 Also referred to as the methylcellulose
assay, is an in vitro assay used in the study
of hematopoietic stem cells.
 The assay is based on the ability of
hematopoietic progenitors to proliferate
and differentiate into colonies in a semi-
solid media in response to cytokine
stimulation.
 The colonies formed can be enumerated
and characterized according to their
unique morphology.
 STEMVISION

36. RNA Isolation
 A single-step method for total RNA isolation from cells and tissues depends
on TRIzol reagent.
 The reagent, a mono-phasic solution of phenol and guanidine isothiocyanate.
 TRIZOL Reagent maintains the integrity of the
RNA, while disrupting cells and dissolving cell
components. Addition of chloroform followed
by centrifugation, separates the solution into an
aqueous phase and an organic phase.
 RNA remains exclusively in the aqueous phase.
After transfer of the aqueous phase, the RNA is
recovered by precipitation with isopropyl
alcohol.

37. Coculture ESCs on S17

38. 40