(Lecture 3) (L3_iPSC.pdf) iPSC

What are the three core components of tissue engineering?

Cells, biomaterial/scaffold, and biochemical factors/molecules

What does ‘pluripotent’ mean in stem cell biology?

Pluripotent cells can differentiate into any cell type derived from the three germ layers, but cannot form an entire organism organism on their own

What is the difference between totipotent, pluripotent, multipotent and unipotent cells?

Totipotent (zygote) → any cell including placenta. Pluripotent (ESC/iPSC) → any somatic cell type. Multipotent (HSC, MSC) → limited set of related types. Unipotent → only one cell type.

Where are embryonic stem cells (ESCs) derived from?

From the inner cell mass (ICM/epiblast) of a blastocyst, typically spare embryos from IVF programmes.

Who first isolated human embryonic stem cells and when?

James Thomson et al. in 1998, published in Science. Cells were derived by plating the isolated inner cell mass on fibroblast feeder layers in the presence of serum.

What are four key limitations of human embryonic stem cells (hESCs)?

1) Immune rejection (allogeneic). 2) Teratoma/tumour formation. 3) May fail to integrate or function fully in host tissue. 4) Ethical concerns around embryo destruction.

What is a transcription factor and what does it do?

A protein that binds regulatory DNA sequences to activate or repress gene transcription. Transcription factors form a complex with RNA polymerase and control which genes are 'on' in a given cell type.

What is the difference between euchromatin and heterochromatin

Euchromatin is diffuse, open chromatin that is actively transcribed (genes 'on'). Heterochromatin is dense, compacted chromatin not involved in RNA synthesis (genes 'off').

What are induced pluripotent stem cells (iPSCs)?

Adult somatic cells genetically reprogrammed back to a pluripotent ESC-like state using specific transcription factors

What are the four Yamanaka factors (OSKM)?

Oct3/4, Sox2, Klf4, and c-Myc. These transcription factors, when introduced into somatic cells, reprogram them to a pluripotent state.

How is pluripotency of iPSCs confirmed experimentally?

Three main tests: (1) embryoid body formation → in vitro differentiation into all three germ layers; (2) expression of ES cell markers (molecular identity); (3) teratoma formation in vivo (tumours containing tissue from all three germ layers).

What is the risk of using retroviral or lentiviral vectors for iPSC reprogramming?

These vectors integrate into the host genome, which can cause insertional mutagenesis, genomic instability, and cancer risk — making them unsuitable for clinical use.

Name two non-integrating viral methods for iPSC reprogramming and their advantage

Adenoviruses (transient DNA, no integration) and Sendai virus (RNA virus, cytoplasmic replication, no genomic integration). Both allow efficient reprogramming without inserting foreign DNA into the genome.

What are three completely integration-free (non-viral) methods for iPSC generation?

1) Episomal plasmids (replicate transiently, no integration). 2) Synthetic mRNA encoding OSKM (very transient, no DNA). 3) Direct protein transduction (no nucleic acid at all).

How do small molecules facilitate iPSC reprogramming? Give two examples.

Small molecules open chromatin, activate stemness signalling pathways, and improve cell survival. Examples: Valproic acid (VPA) and CHIR99021

What is an expression cassette in gene transfer?

The genetic sequence to be delivered — typically a cDNA encoding the gene of interest along with regulatory sequences (promoter, poly-A signal) to control its expression in target cells.

Give one example of a therapeutic application under development using iPSCs.

Generating insulin-producing pancreatic beta cells from patient-derived iPSCs for transplantation in Type 1 diabetes, potentially combined with CRISPR/Cas9 to correct the underlying genetic mutation.

Where are haematopoietic stem cells (HSCs) found and what can they produce?

HSCs are found in bone marrow and blood. They are multipotent and can form all types of blood cells

What is the differentiation potential of mesenchymal stem cells (MSCs)?

MSCs (from bone marrow, adipose tissue, etc.) are multipotent and can differentiate into bone, cartilage, muscle, and fat cells.