Pluripotent Stem Cells

What does pluripotency mean?

The ability to differentiate into any cell type of the three primary germ layers (ectoderm, mesoderm, endoderm)

What are the 2 ways of producing differentiated cells?

Transdifferentiation or pluripotency

What is a challenge of pluripotency?

Difficult to maintain cells in an undifferentiated state.

How are stem cells defined?

They have the capacity to self-renew, differentiate and be clonal

Where are the 5 places stem cells can be found?

Fertilised eggs, early embryos, umbilical cord, adult tissues, cancers.

Why are early embryos artifactual?

The artificial culture conditions and lack of natural developmental signals cannot fully recapitulate in vivo.

Where can stem cells be obtained from adult tissues?

Olfactory bulb (neural), bone marrow (haematopoietic/mesenchymal), intestinal crypts (gut), skin (epidermal/hair/dermal)

Totipotent meaning and example?

Can form all tissues required for reproduction (fertilised egg).

Pluripotent meaning and example?

Had the ability to give rise to all cells of an embryo and adult (e.g., ESCs/iPSCs).

Multipotent meaning and example?

Can form a limited number of cells (tissue stem cells).

Unipotent meaning and example?

Can only form one type of cell (committed progenitor cells).

Self-renewal meaning?

One daughter cell of a dividing stem cell must also be a stem cell.

How are mouse ESCs derived?

From the inner cell mass of a pre-implantation blastocyst E3.5d.

Who defined the culture conditions for mESCs?

Evans and Martin

What growth factor is required to maintain undifferentiated cells?

FGF2

How is pluripotency maintained during self-renewal?

Promotion of proliferation and suppression of differentiation.

What was the original culture method for mESCs?

ICM from diapaused blastocyst cultured on a feeder layer of mouse embryonic fibroblasts (MEFs) with media containing serum to support their growth.

What is a diapaused blastocyst?

The blastocyst comes from an ovariectomised mouse which does not produce oestrogen to prevent implantation in the uterus.

What do MEFs need to be treated with and why?

They need to be treated with mitomycin C to stop proliferation because they grow like weeds and outcompete ICM in culture.

What is mitomycin C?

A DNA alkylating agent that inhibits DNA synthesis to stop proliferation.

What can MEFs be replaced with?

Leukaemia inhibitory factor (LIF) produced by MEFs.

What can serum be replaced with?

Bone morphogenic protein (BMP2/4).

What cytokine family is LIF a member of?

IL-6

Why are MEFs and serum replaced in newer protocols of culturing?

Expensive and introduce batch-to-batch variation.

What family is BMP a member of?

TGFbeta

What 3 pathways does LIF signalling activate?

JAK/STAT, AKT/GSK3B, Ras/Raf/Erk

what is the main way LIF maintains pluripotency in mESCs?

LIF binds LIFr which associates with GP130r (heterodimerisation). JAK binds the phosphorylation docking intracellular domain of GP130r, gets phosphorylated and activated. Activated JAK recruits SH2 domain-containing proteins (STAT3) and phosphorylates it. STAT3 homodimerises and translocates to the nucleus where it binds to promotors of pluripotency genes.

What are the 5 pluripotency genes?

Oct4, Sox2, Nanog, Klf4, c-Myc

How does LIF signalling create a balance between self-renewal and differentiation?

The receptor complex can activate Src family kinases which phosphorylates Shc adaptor protein and Grb2 which recruits and activates Ras/Raf/Mek/MAPK/Erk signalling. Erk can translocate into the nucleus where it activates expression of genes for differentiation.

What is alternative way LIF maintains pluripotency?

The receptor complex can recruit Src/Shc and activate the PI3K/AKT pathway which inhibits GSK3beta. B-catenin stabilises accumulates in cytoplasm and translocates to nucleus to activate expression of Wnt genes involved in self-renewal.

What pathway does BMP4 activate?

BMP4 binds the heterotetrameric complex of type I and type II receptors and type II phosphorylates type I which activates its set/thr activity to phosphorylate SMAD4. SMAD4 translocates to nucleus to activate expression of Id.

What is Id?

Inhibitor of differentiation.

What does Id do?

Increases cyclin D1 to prevent exit from the cell cycle.

What happens in the absence of wnt?

GSK3b is active which phosphorylates b-catenin to mark it for degradation. TCF3 inhibits self-renewal and promotes differentiation.

What happens in the presence of wnt (or AKT)?

GSK3b is inhibited so b-catenin is not phosphorylated for destruction or AKT phosphorylates b-catenin for stabilisation. B-catenin translocates to nucleus to inhibit TCF3 and relieves TCF1 suppression to express c-myc (proliferation)

What is ground state pluripotency?

The naive state that ICM naturally exist in before they receive external differentiation signals (unbiased pluripotency).

How are differentiation signals blocks in vitro?

MEK small molecule inhibitor (Erk pathway) and GSK3b small molecule inhibitor.

What happens to pluripotency under normal conditions?

OCT4 induces expression of FGF4 induces differentiation via MAPK/Erk pathway balanced by LIF/BMP induced self-renewal.

How can you enhance ground state in vitro?

Instead of using LIF/BMP, use small molecule inhibitors to block differentiation instead (3i conditions). Removal of differentiation signals causes natural renewal. Normal external stimuli are despensible.

What are the 3i conditions?

FGFRi (SU54020), MEKi (PD184352), GSK3i (CHIR99021)

What are the culture conditions for mESCs to enter ground state?

LIF + 2i conditions

Who first derived human ESCs?

James Thomson in 1998

What are the ethical and safety issues of hESCs?

Requires use of fertilised eggs from IVF and teratomer risks.

What are the defining features of hESCs?

Self-renewal, indefinite proliferation and pluripotency.

What factors are needed to culture hESCs?

FGF2 and activin A

Why are human and mouse ESCs different?

Represent different stem cell states.

What state do mESCs represent?

Native ground-state early phenotype from E3.5d.

What state do hESCs represent?

Primed epiblast-like pluripotent state from E5.5d.

What are iPSCs?

Specialised cells created from other adult cells through a process of reprogramming.

how can mouse fibroblasts be reprogrammed to embryonic-like pluripotent state?

With the 4 TFs; Oct4, Sox2, c-Myc, klf4.

Why do you need caution with c-myc?

It’s oncogenic so increases the risk of tumour formation.

Why are hIPSC inefficient?

1/5000 cells are reprogrammed to ES-like cells, take 30 days to generate and must seed at low density otherwise get outcompeted.

What are the applications of hiPSCs?

Cell replacement therapies, disease in a dish model, personalised medicine, high-throughput drug screening.

What factors improve the efficiency of iPSC culture?

p53 siRNA and UTF1