STEM Cells (Mostly Key Points)

B4L5

Stem cell

A cell capable of both self-renewal and differentiation into mature cell types

T/F: Pluripotent cells lose their pluripotency over time

True

Pluripotent cells can produce many different cell types but as cells divide they lose their pluripotency and become multipotent, oligopotent, biopotent, and unipotent

Embryonic stem cells

• Easy to establish

• pluripotent and indefinite

• Ethically controversial 

• tumorigenic potential 

Somatic stem cells

• Difficult to establish 

• Multipotent and definite 

• Less controversial 

• Lower potential 

Properties of ES cells

• form and grow in colonies with smooth defined borders

• high ratio of nucleus to cytoplasm and prominent nucleoli

• express pluripotency markers

• high telomerase activity

• Can form chimeras

• Can form teratomas

What’s important to remember about cells on a high passage?

If using cells on passage 60 or a high passage make sure you consider what happened to the DNA at this point, clone frequency can impact experiments in different ways

1st generation Integration based iPSC

• standard moloney viruses (retroviral silencing) 

• Dox-inducible lentiviruses 

• Excisable lentivirus 

• Transposase mediated integration 

2nd and 3rd generation Integration-free (“footprint-free”)

• Adenoviruses

• Sendai virus (RNA-based reproductive cycle)

• Plasmid transfections (regular, minicircles)

• Repeated mRNA transfection

• Self-replicating synthetic RNA (Simplicon)

• Protein transduction, small molecules, miRNAs

Do stem cells proliferate?

No

Totipotency

Can generate the whole organism including embryonic tissues

3 streams of stem cells 

1. Gurdon - Nuclear reprogramming

2. Overexpression of factors in frogs and drosophila

3. Establishing of ES cell cultue

What are the two main capabilities of stem cells?

Capacity to self renew and to differentiate into mature progeny

Ex. Neural stem cells self-renew for many rounds before producing neurons, astrocytes, and oligodendrocytes in the brain

What is the highest level of cellular potency?

Totipotency is the highest level of cellular potency

Totipotency is present only in blastomeres in the 2-4 cell stages of the development before morula formation

How many cell types arise from the blastocyst?

More than 200

What is the relation between the potency level of a stem cell and the number of cell types it can differentiate into?

The potency level of a stem cell is directly related to cell types it can derive

• multipotent hematopoietic cells produce numerous cell types it can derive

• unipotent spermatogonia only produces one cell type - spermatocytes

What are two types of human pluripotent stem cells?

Human embryonic (ESCs) and induced pluripotent stem cells (iPSCs)

• ESCs are derived from the inner cells mass of an early human embryo

• iPSCs are made by reprograming various somatic cell types

What are the main and shared properties of two types of human pluripotent stem cells?

• Both ESCs and iPSCs grow in colonies with smooth borders, have a high nucleus to cytoplasm ratio with very prominent nucleoli 

What are common pluripotency markers that the two types of human pluripotent stem cells share? 

SOX2, OCT4, NANOG, SSEA4, TERT 

What does self renewal of both types of human pluripotent stem cells depend on?

FGF2 supplementation in vivo

Who derived first human embryonic (ESCs) stem cells?

James Thomson established first human embryonic stem cells in 1998

• In subsequent years, his lab defined conditions for growing these cells under xeno-free conditions

Who derived first induced pluripotent stem cells from fibroblasts?

Shinya Yamanaka established first mouse induced pluripotent stem cells in 2006

• Synergy of SOX2, OCT4, KLF4 and c-MYC expressed in mouse fibroblasts led to derivation of cells that in culture exhibited main features of mouse ESCs

• These four transcription factors are collectively called “Yamanaka factors”

What are the three main pluripotency genes?

SOX2, OCT4, and NANOG are key components of the intricate pluripotency network of genes

What reprograming tools are characterized with highest efficiency and safety?

Sendai Virus and mRNA-based tools

• Sendai Virus kit is based on a non-integrating replication deficient RNA virus

• Self-replicating synthetic RNAs (promise of clinical use)