1. EMBRYONIC STEM CELL

3. History…..
■ In 1998, the publication of the generation of human embryonic stem cell (h-ESC) lines contained the statement ‘These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine’. ■ Progress towards the utilization of this technology for regenerative medicine therapies has perhaps not been as rapid, or indeed, as straightforward, as hoped or expected at the time

4. Human embryonic stem cells (h-ESCs)
■ are derived from inner cell mass of blastocyst-stage embryos ■ exhibit unlimited proliferation ability ■ pluripotency to differentiate into various cell types originated from the three germlayers.

5. applied to generate ESC lines in non-human primates and humans.
In mammalian embryonic development, the blastocyst consists of an inner cell layer called the inner cell mass (ICM) and an outer cell layer called the trophoectoderm
(TE).
The successful derivation of a mouse embryonic stem cell line from ICM was first reported in 1981, and subsequently, advances in m-ESC culture techniques were
applied to generate ESC lines in non-human primates and humans.

6. The first stages of development of a fertilized oocyte mouse

10. Human embryonic stem cell lines derived from single
blastomeres

11. h-ESCs
Their unique properties are expected to improve investigations in the field of human development and provide new materials for cell/tissue transplantation therapies and drug screening However, there are prerequisites for clinical application of h-ESCs

12. h-ESCs
■ In general, all processes related to the derivation, maintenance and differentiation of h-ESCs should be accomplished under xeno-free culture condition using good manufacturing practice (GMP) systems
■ Guided-differentiation protocols of h-ESCs into a given functional cell type must be established so that the resulting cells are homogeneous and do not form teratomas or cause cancer, and immune responses/rejection caused by the transplantation of h-ESCs or their differentiated derivatives should be prevented

13. h-ESC Derivation The most important process involved in hESC
derivation might be the isolation of the ICM from the TE.
In the initial stages of h-ESC research, immunosurgery
procedure employing anti-human serum and guinea pig
complement was used for isolation of the ICM.
However, these reagents may include animal pathogens
and molecules, and the exposure of animal-derived products to the ICM and its derivatives, h-ESCs, during immunosurgery may bring about immune responses in patients after transplantation.

14. h-ESC Derivation To avoid the contamination of
animal-derived components during the derivation procedure, several methods have been used instead of immunosurgery:
Mechanical dissection and Mechanical isolation
of the ICM using flexible metal needles with sharpened tips has been introduced as a method for h-ESC derivation.

15. h-ESC Derivation
■In the first report of h-ESC derivation in 1998 mouse embryonic fibroblast (MEF) cells were used as feeder cells to support the undifferentiated proliferation of hESCs, and leukemia inhibitory factor (LIF) and fetal
bovine serum (FBS) were used as major components of the
culture medium.
■ However, the bovine serum generally
used in cell cultures is a complex mixture of proteins of
unknown composition and, is sometimes associated with
batch-to-batch variation. Serum may also contain factors
inducing h-ESC differentiation.
■ To obtain stable and reproducible
results, studies have been performed to identify
essential serum-provided components in various stem
cell cultures.

16. MEF-conditioned medium on extracellular matrix-coated Dishes.
h-ESC Derivation
■ KnockOut Serum Replacement (KO-SR), which is a commercially available serum substitute, has been developed and contains components such as bovine albumin, transferrin and insulin. The proliferation of h-ESCs cultured in KO-SR containing media
can be successfully maintained in undifferentiated state, and soon after this substance was developed, animal serum was replaced by KO-SR for h-ESC culture
■ LIF, which is a key regulatory factor involved in m-ESC self-renewal via the JAK/STAT3 signaling pathway in feeder-free systems, does not have the same effect in h-ESCs. It was found that h-ESCs could be grown in feeder-free conditions using
MEF-conditioned medium on extracellular matrix-coated Dishes.
■ Based on the studies for h-ESC culture conditions, basic fibroblast growth factor (b-FGF) is the most important factor for maintaining h-ESC pluripotency and, is now commonly used in h-ESC culture medium, both in the presence and in the absence of feeder cells

17. h-ESC Derivation
■ In h-ESC cultures, feeder cells provide both a suitable attachment substrate and important soluble factors for the maintenance of undifferentiated
state. Various human feeder cells that originate from fetal skin, foreskin, fallopian tubes, uterine endometrium, bone marrow, amniotic epithelium and other tissues have replaced MEFs in h-ESC culture
■ Based on the supportive ability of h-ESCs as fibroblast-like cells for maintaining the undifferentiated growth of h-ESCs in feeder- free conditions, several studies have shown that h-ESC derived fibroblast cells can be used as feeder cells for h-ESC culture. It was recently reported that immortalized
human foreskin fibroblasts induced to secrete b-FGF by lentiviral transfection could be used for reproducible and cost-effective culture of h-ESCs
■ To eliminate the need for feeder cells and establish defined culture conditions for h-ESC growth, various coating materials to support the attachment of h-ESCs and supplementary factors in h-ESC culture medium have been studied. Matrigel, a complex mixture including extracellular matrix molecules, several growth factors and other substances, has been commonly used as a coating material for h-ESC culture

18. h-ESC Derivation
■ To replace Matrigel with more defined substrates, several research groups have tested laminin, fibronectin, collagen I, collagen IV, vitronectin and human serum as alternative substrates for feeder cells both alone and in combination for h-ESC cultures
■ Several cytokines and/or growth factor(s) have been tested for their ability to support undifferentiated growth of h-ESCs together with
B-FGF under feeder-free culture conditions. Stimulation of MEFs with b-FGF ultimately results in the regulation of key members of the
transforming growth factor beta (TGFβ) pathway, including
up-regulation of Inhba, TGFβ1 and Grem1 and
down-regulation of BMP4.
■ Other factors expected to play a role in maintaining pluripotency are the insulin-like growth factors (IGFs), based on a report that inhibition of the IGF1 receptor represses h-ESC self-renewal, while the addition of an IGF1 analog facilitates the expansion of undifferentiated cells

19. h-ESC Derivation
■ The Wnt signaling pathway is also thought to affect h-ESC self-renewal, but its role is currently unclear. It was reported that the addition of Wnt1 to MEF-conditioned medium represses spontaneous differentiation of h-ESCs, whereas Wnt3a has been reported to induce h-ESC differentiation. Additionally, it was shown that low concentrations of glycogen synthase kinase-3 (GSK-3) inhibitor induce self-renewal, but higher concentrations promote mesoderm differentiation
■ With regard to clinical applications, a defined feeder-free culture medium with only human-originated proteins/components should be developed for hESC culture, and furthur efforts to determine the factors responsible for hESC self-renewal and pluripotency are required.

20. h-ESC Derivation
■ In 2006, a defined feeder-free culture medium for h-ESCs (TeSR1) composed of a DMEM/F12 base supplemented with human serum albumin, vitamins, antioxidants,
trace minerals, specific lipids and cloned growth factors was developed, and new h-ESC lines were established in these culture conditions.
■ The finding of this study suggested that the addition of b-FGF, LiCl, γ-aminobutyric acid (GABA), pipecolic acid, and TGF-β into a defined culture medium supported undifferentiated growth of h-ESCs.
■ Because the cost of this defined medium makes it difficult to use routinely, a modified version (mTeSR1) that includes the use of animal source proteins (bovine serum albumin and Matrigel) and cloned zebrafish b-FGF has been developed .

21. How to maintain ES cells in an undifferentiated state
Mouse ES cells
■ LIF
■ Culture medium enriched of serum (10%-15%)
■ Fybroblasts: feeder layer
■ Plastic colture types
■ % CO2
Human ES cells
■ No LIF, KSR + feeder+ b-FGF
■ Matrigel, no feeder but specific medium

22. Embryonic stem cell colony
Neural differentiation from human embryonic stem cells. 
Staining: neural marker TUJ1 (green) and DNA (blue).

23. Martin John Evans, a british scientist, credited with discovering how to culture embryonic stem cells in 1981, and for his work in the development of the knockout mouse and the related technology of gene targeting. In 2007, he was a co-winner of the Nobel Prize in Physiology or Medicine in recognition of his gene targeting work.

24. In 1998, Thomson’s Lab was the first to report the successful isolation of human embryonic stem cells. On November 6, 1998, Science published this research in an article titled "Embryonic Stem Cell Lines Derived from Human Blastocysts", results which Science later featured in its “Scientific Breakthrough of the Year” article, 1999.

25. ES Advantages ES Disdvantages
■ Source
■ Potential for a wider variety of applications than do adult stem cells.
■ Proliferation ability and maintenance of the undifferentiated state
ES Disdvantages
■ Source: Ethical issues
■ Mixed cell populations
■ Teratomas formation in vivo

26. ES isolation
■ They can be extracted from the embryo at the blastocyst stage and grown in the laboratory in the form of cell lines that multiply indefinitely.
■ Only in 1998 it has instead managed to derive stem cells from human embryos created in a test tube with artificial insemination and donated for research.
■ Embryonic stem cells can also be derived from embryos obtained by the technique of cloning or nuclear transfer.

27. ESC sources: ESCs can be isolated from fresh, frozen, dead, excess and genetically deficient embryos, by parthenogenesis and somatic nucleus transfer, from biopsies, and from pluripotent stem cells obtained from adult tissues.

28. ES cells: a valuable tool for research and not only ...

29. Nuclear transfer by microinjection

30. The discovery of iPS cells
In 2006, Shinya Yamanaka made a groundbreaking discovery that would win him the Nobel Prize just six years later: he found a new way to ‘reprogramme’ adult, specialized cells to turn them into stem cells.
These laboratory-grown stem cells are pluripotent – they can make any type of cell in the body - and are called induced pluripotent stem cells, or iPS cells.
Only ES cells are naturally pluripotent: Yamanaka’s discovery means that theoretically any dividing cell of the body can now be turned into a pluripotent stem cell.

31. So how are these iPS cells made
So how are these iPS cells made? Yamanaka added four genes to skin cells from a mouse. This started a process inside the cells called reprogramming and, within 2 – 3 weeks, the skin cells were converted into induced pluripotent stem cells. Scientists can now also do this with human cells, by adding even fewer than four genes.

36. ES cells: a valuable tool for research and not only ...

37. Study of the function of a gene “in vivo”
Chimeric mice production: the most frequently used approach is blastocyst microinjection. By injecting gene-targeted ES cells into the cavity of blastocyst (i.e. 3.5-dpc embryo), researchers can get chimeric mice which is originated from the ES and host blastocyst.

39. knock-out p63

40. c-myc mutant
Wild type
c-myc mut

41. ADAM17 mutant

42. ES cells: a valuable tool for research and not only ...

43. The differentiation potential of ES

44. Main Methods of ES differentiation
Embryoid Bodies formation
Growth on stromal cells
Growth on extracellular matrix protein

49. Current strategies by which pluripotent cells ESCs or iPSCs can be converted into any number of cell fates by pushing the cell through a series of intermediate stages similar to those that occur during in vivo development. At each stage, growth factors and small molecules can be used to push the differentiation of the cells towards the desired fate. At each stage, a representative marker used to identify cells is noted, although generally multiple markers are used to analyse the desired cells. Below the final stage, the approximate percentage of cells that achieve the desired fate, based on the analysis of different markers, is indicated

50. Differentiation in nerve cells
ES undifferentiated
+RA
no siero
EBs
Monolayer
Neurons
Glia cells

51. Formation of insulin-producing cells (IPCs)
A: Embryoid body formation
B: Definitive endoderm formation

52. ES cells: a valuable tool for research and not only ...

53. ES cells: a valuable tool for research and not only ...

54. The use of human ESCs as resource for cell therapeutic approaches is currently performed for several diseases

55. Parkinson disease

56. Actor Michael J. Fox, affected by Parkinson's disease at the age of thirty years. It is probably the most famous supporter on the use of stem cells in the treatment of this disease and created a foundation (the Michael J. Fox Foundation) which raises money for research into new treatments for Parkinson's.

57. Reeve, James Thomson and John Gearhart
■One of the major supporters of embryonic stem cells was actor Christopher Reeve, paralyzed from a riding accident.
Reeve favored stem cell research as a key to regenerating spinal cord and other nervous system tissue.
■ He had also been submitted a transplant of stem cells that unfortunately it gave poor results.
■ Even following his commitment, USA public opinion is largely favorable to the use of embryonic stem cells to cure serious diseases
Reeve, James Thomson and John Gearhart

58. In 2010 the Food and Drug Administration (FDA) has issued the first permits for carry out experiments on humans with embryonic stem cells, marking a turning point for the development of future therapies with these cells: experiments designed to repair spinal cord injuries in trauma victims and to rebuild the retina in patients with eye diseases.

59. The California-based biotech company Geron was at the vanguard with an hESC-derived oligodendrocyte progenitor cell product, GRNOPC1, as a treatment for spinal cord injury.
As part of a phase 1 safety study, the first patient was dosed around 13 years after the initial h-ESC line derivation paper.
Unfortunately, only five subjects were dosed before the trial was halted though for strategic rather than safety reasons.
Interestingly, Asterias Biotherapeutics has now licensed the technology and is re-initiating a phase 1 clinical trial

64. At the end of 2014, the first hESC-derived cardiac cell implant was performed by Prof. Menaschè and his team in a woman of 68 years suffering from severe heart failure. Ten weeks after the intervention, the patient is feeling well, her condition has improved markedly, with no complications having been observed.

65. Indeed, their hESC-derived retinal pigment epithelial (RPE) cell therapy product MA09-hRPE has been dosed to 18 subjects in Europe and the USA with either Stargardt’s disease or age-related macular degeneration (AMD), and shown to be generally safe and well tolerated. A recent report has also described the dosing of four Asian patients with MA09-hRPE.

67. International Human Embryonic Stem Cell Laws
Countries around the world have responded to the ethical problems
raised by embryonic stem cell research in a number of ways. Some governments have passed laws prohibiting all research on human embryos,
while others have explicitly endorsed and funded ES cell research.

70. Religious debate over h-ES cells Pro-choise group believe that:

71. Religious debate over h-ES cells Pro-life group believe that: