The document discusses the benefits of exercise for mental health. Regular physical activity can help reduce anxiety and depression and improve mood and cognitive functioning. Exercise causes chemical changes in the brain that may help protect against mental illness and improve symptoms.

1. Pluripotent stem cells (PSCs):
Applications in
disease modeling, drug discovery and
regenerative medicine
TARA SINGH RAWAT
Ph.D. Scholar
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
1

2. Content:
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
2

3. INTRODUCTION
• Experimental modeling of human disorders gives a comprehensive view of the biological processes
underlying human pathologies which is crucial to devising strategies for their prevention and
treatment.
• The properties and applications of different types of pluripotent stem cells (PSCs) have received an
incredible degree of attention in recent years, which was difficult to imagine during the first attempts
to move into this area.
• The discovery of induced pluripotent stem cells (iPSCs) has opened up unprecedented opportunities in
the pharmaceutical industry, in the clinic and in laboratories. In particular, the medical applications of
human iPSCs in disease modeling and stem cell therapy have been progressing rapidly.
• The ability to model human diseases using cultured iPSCs has revolutionized the ways in which we
study monogenic, complex and epigenetic disorders, as well as early- and late-onset diseases, creating
new possibilities for the establishment of therapies.
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
3

4. 6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
4
HISTORY
Time-line for 100 years
of research on stem
cells with
scientific breakthroughs
(Danish society of stem cell, 2016)

5. Timeline of the key dates in the development of pluripotent stem
cell-based therapies
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
5
HISTORY- cont..
(Erin et al., 2015)

6. Pluripotent Stem cell as a Research Tool
Indefinite
capability
to divide
Self renewability and high potency of
pluripotent stem cell makes these cells more
reliable tools over animal model
CELL
POTENCY
Decreasingcellpotency
Totipotent cell (Morula)
Oligopotent cell
Unipotent (germ cells)
STEM
CELL
SELF
RENEWAL
Pluripotent cell (Blastocyst inner cell mass)
Easeinhandlingin-vitro
Indefinite
capability
to divide
Indefinite
capability
to divide
Indefinite
capability
to divide
Capability
to
Differentiate
Asymmetric
division
Multipotent cell (bone marrow cells)
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
6

7. Why Pluripotent stem cell??
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
7New York Stem Cell Foundation, Webinar,2015

8. Importance of human Pluripotent Stem cell (hPSCs)
Limitations of animal model
1. Difficulties in recapitulation of human disease phenotypes in animal model.
eg. mutations in the BLM gene in humans lead to Bloom syndrome, but in its orthologue in mouse are fatal.
2. Translational difficulties
eg. Drug tested in animals can not be effective in human.
Therefore it is preferable to conduct experiments in human.
Three major advantages:
1. hPSCs are normal primary cell lines.
2. have an intrinsic capability for indefinite self-renewal.
3. have the potential to adopt virtually any cellular fate through differentiation.
These properties enable us to study genotype–phenotype relationships in a broad range of
human cell types and differentiation states, as well as to obtain large numbers of cells for
drug screening and cell therapy.
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
8

9. Types of Pluripotent Stem Cell used for disease modeling and
therapeutic applications
Stem cells can be derived from human embryos or somatic tissues or they can be
created by inducing greater potency in an differentiated somatic cell.
• Embryonic stem (ES) cells
• Nuclear Transfer ES cells
• Induced pluripotent
stem cells (iPSCs)
(New York Stem cell Foundation)
(Ankshita et al., 2015)
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
9

10. Impact of reprogramming on Disease study
Histcompatible Medium Availability
High proliferation
Pluripotency
High Availability
High proliferation
Pluripotency
Histocompatible
Isolation
Heterogeneity
Limited Expansion
Cellular fitness
Low potency
Teratomas
Histoincompatibility
Social issues
Ethical issues
Teratomas
Cons
Pros
Adult Stem Cell ES Cells iPSC Cells
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
10

11. Potential Applications of Patient-Specific hPSCs
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
11
Sayed et al., 2016

12. Generation of disease models using human PSCs
• All studies utilizing human PSCs for disease modelling begin by
establishing cell lines carrying the molecular defects of interest.
• These cells are then used to identify a robust disease phenotype in
either the undifferentiated or differentiated state.
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
12

13. Strategies for generating disease models using human pluripotent stem cells (PSCs)
6/21/2018 13Yishai et al., 2016Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur

14. Large-scale initiatives for derivation of diseased iPSCs
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
14
The goal of these repositories is to provide cell lines for most genetic disorders for
research Yishai et al., 2016

15. Identifying disease-related phenotypes
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
15
Modelling human diseases in vitro requires the identification of clear, disease-
specific phenotypes in differentiated as well as in undifferentiated state
Cellular
phenotypes
Physiological
phenotypes
Molecular
phenotypes
Cultured iPSCs
Yishai et al., 2016

16. Modelling different disorders with human PSCs
Several criteria should be taken into account when choosing a stem cell-based model
for the study of human diseases
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
16
Yishai et al., 2016

17. 6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
17

18. Published modeling of blood diseases using human patient-specific
iPSCs
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
18

19. Drug screening using iPSCs
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
19

20. Drug screening approaches using iPSCs
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
20
Yishai et al., 2016

21. Conventional Versus iPSC-Based Drug Discovery
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
21Sayed et al., 2016

22. Drug screening for neurological diseases using patient-
derived induced pluripotent stem cell models
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
22
Yishai et al., 2016

23. Cont…
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
23
Yishai et al., 2016

24. The bench to bedside pathway
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
24

25. Leading Applications of Pluripotent Stem Cell Derivatives
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
25Alan et. al., 2016

26. ESC Trials for Cell therapy
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
26Alan et. al., 2016

27. hPSCs in Regenerative Medicine
• Process of REPLACING, ENGINEERING or REGENERATING human cells, tissues
or organs to restore or establish normal function.
• Engineering damaged tissues and organs via stimulating the body's own repair
mechanisms to functionally heal previously irreparable tissues or organs.
• Growing tissues and organs in the laboratory and safely implanting.
• Thus hPSCs are indispensable to the success of regenerative medicine.
(Chris Mason et al., 2008)
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
27

28. 6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
28
in vitro grown beating human heart cell
in vitro cultured beating human heart cells

29. 6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
29
• Report the safety and tolerability
of subretinal transplantation of
human embryonic-stem-cell
(hESC)-derived retinal pigment
epithelium
• Followed for 1 year there was no
evidence of adverse proliferation,
tumorigenicity, ectopic tissue
formation, or other serious safety
issues related to the transplanted
cells
Colour fundus photographs of a patient with Stargardt’s macular dystrophy
(dotted circle shows an outline of the transplanted area).
G- A large central area of atrophy is visible on the preoperative photograph
H -An area of transplanted retinal pigment epithelium cells is visible at the superior
half of the trophic lesion
I- at 6 months that becomes larger and more pigmented at 15 months

30. Generation of human liver buds from human iPSCs6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
30

31. The bench to bedside pathway
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
31
Rebuilding heart with patients cells
(Brendan, 2013)

32. Basic idea behind engineering a organ
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
32
THE SCAFFOLD
Decellurization
THE CELLS
Recellurization with patients cells THE BEAT
(Brendan, 2013)6/21/2018

33. Stripping donor hearts and repopulating them with recipient-derived
stem cells
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
33
perfusion decellularization setup
Jacques et. al., 2015
A human heart before (i), during (ii), and after (iii)
pressure-controlled perfusion
i
ii iii
A decellularized human heart
awaits rebuilding
with an injection of precursor
cells.
partially recellularized human whole-heart
bioreactor
6/21/2018

34. Synthesizing Kidney with 3d printer
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
34
Anthony Atala: Printing a human kidney | TED Talk | TED.com
A 3-D printer lays down scaffolding and cells to
create the beginnings of a kidney.

35. Rebuilding Heart Valve in vitro
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
35

36. 6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
36
Engineered human
bladder In growth
solution

37. Case study
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
37

38. INTRODUCTION
• Diabetic cardiomyopathy is a complication of type 2 diabetes,
with known contributions of lifestyle and genetics which
progresses to dilated cardiomyopathy and heart failure.
• The World Health Organization estimates that the lethal clinical trajectory of type 2
diabetes mellitus (T2DM) will make it the seventh leading cause of death worldwide by 2030.
• Till date no specific drug treatment exists to prevent diabetes-induced cardiac dysfunction
• Induced pluripotent- stem-cell-derived cardiomyocytes used to develop environmental and
patient-specific in vitro models recapitulating the condition.
• These models are harnessed in a phenotypic screening assay that identifies candidate
protective molecules.
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
38
Heart affected with cardiomyopathy

39. Basic Strategy
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
39

40. Experimental Procedures
1. Cadiomyocytes(CM) generation and maturation from iPSCs
2. Induction of Diabetic Cardiomyopathy in iPSCs-CM
3. Immunofluorescence assays
4. Measurement of CM protein secretion
5. Cardiomyocyte score analysis
6. Small molecule screening
7. Electron microscopy
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
40

41. Results
in vitro Maturation of iPSC-CMs. (SM- standard maintenance media, MM- Maturing media)
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
41
Metabolic Manipulation of iPSC-CMs Enhances Characteristics Associated with Maturing CMs
(A) alpha-actinin (green); DAPI (blue)
(B) CM score following exposure to standard medium (SM) or maturation medium (MM) for 3
days.
(C) Sarcomere length in CM exposed to SM or MM for 3 days. a.u., arbitrary units.

42. A Diabetogenic Extracellular Environment Induces Cardiomyopathy in
mature iPSC-CM cells in-vitro
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
42
A&B- Increased BNP level in response to DM ) (DM- Diabetogenic Media)
D- CM score was diminished after 2 days exposure to DM .
E- increases in cellular and nuclear surface area definitive confirmation of hypertrophy
C&F- The regular striped pattern of a-actinin staining became diffuse.
loss of sarcomeric integrity

43. Diabetic-Patient-Specific iPSC-CM Exhibit a Diabeto Mimetic
Phenotype in-vitro
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
43

44. 6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
44
High-Content Phenotypic Screening Identifies Small Molecules
that Can Prevent Development of DCM in vitro
MM-maturing media, mDM- modified Diabetogenic media
BM- A combination of 10 mM Mifepristone and 100 mM Bosentan effectively prevented onset of the
diabetic phenotype (B– D).

45. 6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
45
Pie chart to show the functional categories
of positive compounds identified in the primary
screen. Compounds were categorized according
to their primary mode of action, and examples of
the chemical structure of selected molecules from
each category are shown.
Pie chart showing the functional categories of positive compounds identified in the primary
screen. Compounds were categorized according to their primary mode of action, and examples of
the chemical structure of selected molecules from each category are shown.

46. The Phenotype of Patient-Specific Diabetic CMs can Be Rescued
Pharmacologically
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
46
For both cell types, the most-effective compounds were Thapsigargin and Fluspirilene

47. SUMMARY
• Diabetic cardiomyopathy can be induced in vitro by environmental or genetic
means
• Diabetic patient-specific cardiomyocytes show baseline cardiomyopathy
• The extent of patient-specific cardiomyopathy is clinically correlated
• Phenotypic screening identifies drugs that rescue the disease phenotype
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
47

48. CONCLUSION
• Comprehensive understanding of disease etiology is the key to design
strategy for effective treatments of diseases.
• The derivation of hPSCs especially iPSCs has transformed modern biology
with their abilities to indefinitely expand in vitro and generate all adult
lineages in vivo, combined with gene editing technologies, have provided us
with a vast treasure of human disease models.
• Thus pluripotent stem cell especially iPSCs are key tools for research and
development in disease modeling, drug screening and regenerative
medicine.
6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
48

49. 6/21/2018
Department of Plant Molecular Biology and Biotechnology,
IGKV, Raipur
49