DERIVATION AND DIFFERENTIATION OF

PLURIPOTENT STEM CELL AND

INDUCED PLURIPOTENT STEM CELL

K.MOHANYA
PRK20BT1023
M.TECH BIOTECHNOLOGY
 History of Pluripotency
• 1961 (Till and McCulough) - Multipotent stem cells discovered.
• 1998 (Thomson et al) - Human PSCs isolated.
• 2005 (Dvorak et al) - Role of FGF2 identified in preserving undifferentiated states.
• 2006 (Takahashi and Yamanaka) - Human PSCs isolated.
• 2007 (Takahashi et al, Yu et al) - Human iPSCs generated from fibroblasts using retrovirus and lentivirus.
• 2008 (Huangfu et al) - Small molecules, e.g. valproic acid, enhance iPSC generation.
(Okita ) - Human iPSCs generated using non-integrating plasmids.
• 2009 (Kaji et al, Woltjen et al) - Self-excising vectors, e.g. piggyBac transposon, can generate human iPSCs.
• 2010 (Warren et al) - Synthetic mRNA delivery generates iPSCs.
• 2011 (Karow et al) - Cre-loxP excision generates non-integrating human iPSCs.
• 2012 - Nobel prize awarded to Shinya Yamanaka for iPSCs
• 2013 (Hou et al) - Complete chemical induction (e.g. valproic acid, CHIR99021, FSK) of mouse
fibroblasts into iPSCs.
• 2014 (De Bonis et al) - Role of SIRT1 determined in telomere elongation and genomic stability of iPSCs.
• 2015 (Faulkner-Jones et al) - Bioprinting of human iPSCs.
• 2016 (Hockemeyer and Jaenisch) - Use of CRISPR technology in iPSCs.
• 2017 (Mandai et al) - iPSC-derived retinal cells transplanted in woman suffering from advanced macular
degeneration
 Pluripotent stem cells
• The stem cells was first described by Drs. James A. Till and Ernest A. McCulloch at the University of Toronto
in Canada .
• They found that stem cells derived from mouse bone marrow cells had the ability to differentiate into a
variety of cell types, and were thus called pluripotent stem cells (PSCs).
• PSCs are characterized by the properties of self-renewal and potency.
• 2 main types types of PSC – ESCs and iPSCs.
Derivation of human PSCs and iPSCs
Differentiation of human PSCs
A scheme of the generation of induced pluripotent stem (IPS) cells. (1) Isolate and culture donor cells. (2) Transduce
stem cell-associated genes into the cells by viral vectors. Red cells indicate the cells expressing the exogenous genes.
(3) Harvest and culture the cells according to ES cell culture, using mitotically inactivated feeder cells (lightgray). (4) A
small subset of the transfected cells become iPS cells and generate ES-like colonies.
1. Integrative viral transfection- Retroviruses and lentivirus
2. Non- Integrated Reprogramming vectors
• mRNA reprogramming is very efficient, requires the least amount of patient material, works particularly
well with skin cells from very young donors, and never results in retention of the delivery vectors. But it is
relatively labor-intensive and cannot reprogram blood cells.
• Sendai-viral reprogramming is less technically demanding and more reliable than mRNA reprogramming.
But it is costly; the vectors are eliminated only slowly; and it is not yet amenable to clinical adaptation.
• DNA episomes can reprogram many cell types, and are easy and inexpensive to prepare in both research
and pharmaceutical settings. Like Sendai-viral reprogramming, episomal reprogramming is minimally labor
intensive, and, like mRNA reprogramming, it does not utilize viral derivatives that might harm patients.
However, there is risk that cells will misidentify episomes as broken pieces of their own DNA and splice
them into their own genomes. These rare genomically-integrated cells are already identified and removed
as part of the episomal reprogramming workflow, but in the clinic, where time is often of the essence, this
technique may not always be feasible.
• Self-excising vectors- Cre-lox, Piggy-Bac transposon
• Non-integrative, non-viral vectors- Valproic acid, miRNA, siRNA
 In vitro vs In vivo reprogramming
• Specific types of desired cells may also be obtained directly in vitro or in vivo without a stem cell
reprogramming process.
• The combination of three factors genes, Ascl1, Brn2, and Myt1l, was shown to be sufficient to induce rapid
transformation of in vitro mouse ESCs and postnatal fibroblasts into functional neurons.
• The second-generation cellular reprogramming by way of direct in vivo approaches, which may overcome
critical challenges associates with in vitro systems such as contamination, and time-intensive processing .
• This approach relies on the native micro-environment to produce natural products and obtain in situ
recovery of locally degenerated and damaged tissues.
• Zhou et al.(2008) reported on the in vivo reprogramming of pancreatic exocrine cells into beta cells with the
transcription factors NGN3, PDX1, and MAFA
• In addition, use of the transcription factors FOXa3, GATA4, HNF1a, and HNF4a generated hepatocyte-like
cells directly from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis in vivo, suggesting this
approach may lead to treatment for chronic liver disease.
 Inducing pluripotency with genomic modifications
• Yamanaka factors - Oct4, Sox2, Klf4 and c-Myc (OSKM)
Necessary to reprogram mature mouse fibroblasts into ESC like state, creating iPSC.

• Yu and colleagues - Modified four factors - Oct4, Sox2, Nanog and Lin28
Efficiency similar to that Yamanaka factors.

• Feng and colleagues- Three factor method – Esrrb, Oct4 and Sox2
Mouse embryonic fibroblasts (MEFs) was differentiated into iPSCs with better proficiency than yamanaka
factors

• Two factors combination of Oct3/4, Sox2, Klf4 and c-Myc

• Single factor used Oct3/4

 Promoting iPSC pluripotency with molecules and genetic signaling
• Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling is important
for cell cycle progression, proliferation, and differentiation, and also contributes to carcinogenesis.
• ERK interventions have had seemingly paradoxical effects on stem cells.
• That is, the activation of ERK signaling has been shown to support maintenance of mouse ESC pluripotency;
conversely the inhibition of MEK/ERK signaling with a MEK (MAPK/ERK kinase) inhibitor has also been
shown to support self-renewal and pluripotency of mouse ESCs.
Induction and enhancement of cell reprogramming by RNA signaling
Micro RNAs plays a critical role in
stem cell reprogramming and
maintainance
 Inducting and enhancing pluripotency in iPSCs using chemicals
• The classic targets for these molecules are growth factor receptors or their associated downstream kinases
that regulate intracellular signalling pathway during differentiation.
• Example
1. Sodium butyrate – stimulates histone H3 acetylation, DNA methylation and expression of
pluripotency associated genes.
2. Histone deacytelase inhibitors - Trichostatin A - upto 15 fold increase in efficiency with OSKM.
- Suberoylaniline hydroxamic acid - apprx 2 fold increase
Influences of engineered biochemical and biophysical cues on iPSC preparation efficiency
Cell culture, medium, and material for iPSCs growing environment
 iPSC expansion
Targets, 2D modified methods and practical difficulties of iPSC expansion

• It is generally recognized that ∼10^8 cells are needed for micro-tissue construction in vitro and 10^9 –10^10
cells for repairing solid organ defects in vivo .
• For example,
- 6 × 10^8 β cells are needed for insulin independence after pancreatic islet transplantation
- 1–2 × 10^9 cardiomyocytes for cardiac regeneration after myocardial infarction
- 1.2 × 10^9 CD34+ cells for reconstituting stable blood formation after chemotherapy or irradiation
treatment

• Feeder-free culture systems coated with various ECM proteins were developed to replace feeder-based culture
systems to keep the biochemical components more tightly controlled.
• Among these, ECM proteins such as recombinant vitronectin and laminin successfully replaced the
heterologous Matrigel for hiPSC culture.
• Surface chemistry methods have been utilized to synthesize biomaterial-based substrates or matrices to
harness or emulate the cell– matrix interactions occurring within the native stem cell microenvironment for
better cell adhesion, maintenance6 and expansion.
Common 3D engineering-derived approaches for iPSC expansion

1. Gel embedding
In 2013, Lei and Schaffer were able to obtain ∼20-fold expansion of hiPSCs per passage after only 4–5 days in
culture using a thermoresponsive Mebiol Gel embedding.
And this chemically defined culture system was proved capable of 10^72-fold expansion after 60 passages in as
long as 280 days, while maintaining high pluripotency.

2. 3D scaffolds
Different materials have been used to build scaffolds for stem cell culture.
Different inoculation effects can be achieved depending on the method used to culture stem cells on 3D
scaffolds

3. Microcarriers
Solid microbeads with typical diameters between 40 and 120 μm formed by biomaterials such as glucose,
gelatin or collagen.
20-fold expansion of iPSCs can be attained in a 7 day culture using Matrigel coated DE-53 cellulose
microcarriers and stirred spinner flasks.
 4. Microencapsulation
The process by which cells, bioactive substances and culture media are embedded together into gels or
semi-permeable membranes to form microcapsules, microfibers or microtubes with diameters ranging from
approximately 0.7–1.5 mm.
Microcapsules can provide enhanced growth conditions and protection for the cells contained inside, from
the adverse effects of agitation related shearing actions.

5. Bioreactors
Bioreactors can keep cells in a state of suspension with ideal concentrations of dissolved oxygen and
nutrients to improve cell expansion.
Various kinds of commercial bioreactors have been used for iPSC culture, including stirred suspension
bioreactors, spinner flasks, Erlenmeyer flasks and suspension culture plates, that contained iPSCs in the form of cell
aggregates, microcarriers or microcapsules.
 Engineering-derived approaches for iPSC cryopreservation
• Cell cryopreservation is essential during the process of scalable expansion.
• Poor cryopreservation is associated with low cell recovery rates and reduced pluripotency and thus
necessitates even higher cell yields during iPSC preparation and iPSC proliferation
• Microencapsulation can be used to improve cryopreservation of iPSCs.
• hPSCs embedded inside microcapsules had survival rates above 70% one day after thawing from
cryopreservation in liquid nitrogen. In addition, these cells showed higher metabolic activity than those
thawed from normal cryopreservation without encapsulation.
 APPLICATION

Human development Disease modelling and Cell therapy

drug discovery
Disease modelling and drug discovery
 Approved Clinical Trials for
Pluripotent Stem Cell-based
Products

a- Trial currently halted due to

financial reasons.

https://www.clinicaltrials.gov/ct2/ho
me
Home - ClinicalTrials.gov
 Clinical trials of induced pluripotent stem cells
• Completed – 23
• Recruiting- 39
• Enrolling by invitation- 4
• https://clinicaltrials.gov/ct2/home
• As of today, 53 clinical trials are in progress for various disease indications using iPSCs.
• Neurological diseases have attracted a lot of attention, representing 23% of the ongoing
clinical trials. Clinical trials for the treatment of cardiovascular diseases account for 19%.
• The third largest group of diseases using iPSCs in clinical trials is ophthalmologic diseases,
accounting for nearly 15%. The other disease categories include respiratory, cancer, genetic,
metabolic and other applications.

Induced Pluripotent Stem Cell Clinical Trials, by Disease and Country (bioinformant.com)
• Several drug candidates using iPSCs for screening are in clinical trials in the U.S., France,
Germany, Israel, Taiwan and China.
• Currently, there are studies underway involving iPSC-derived therapeutics for the
treatment of cardiovascular, neurological and metabolic disorders, as well as the repair
of damaged cartilage, spinal cord, motor neurons and eye tissue resulting from genetic
causes or injury.
• While Japan is conducting a large amount of iPSC-research in human patients, it is using
the format of physician lead studies.
 REFERENCE
• Bar, S., Benvenisty, N. Human pluripotent stem cells: derivation and applications. Nat Rev Mol Cell
Biol (2020). https://doi.org/10.1038/s41580-020-00309-7
• Yang Li et al, Engineering-derived approaches for iPSC preparation, expansion, differentiation and
applications. Biofabrication (2017)9 032001. https://doi.org/10.1088/1758-5090/aa7e9a
• Liu, G., David, B.T., Trawczynski, M. et al. Advances in Pluripotent Stem Cells: History, Mechanisms,
Technologies, and Applications. Stem Cell Rev and Rep 16, 3–32 (2020). https://
doi.org/10.1007/s12015-019-09935-x
• Julia Ye and Robert blelloch, Regulation of Pluripotency by RNA Binding Proteins, Cell Stem Cell,
Volume 15, Issue 3, 2014, Pages 271-280, ISSN 1934-5909, https
://doi.org/10.1016/j.stem.2014.08.010.
• Qintong Li, Richard I. Gregory, MicroRNA Regulation of Stem Cell Fate, Cell Stem Cell, Volume 2, Issue
3, 6 March 2008, Pages 195-196. https://doi.org/10.1016/j.stem.2008.01.016
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