La cellula staminale

A life story…

Cellula capace di dare origine a tutte le popolazioni cellulari di uno o più tessuti.

stem cell

What is a stem cell?

stem cell

SELF-RENEWAL(copying)

specialized celle.g. muscle cell, nerve cell

DIFFERENTIATION(specializing)

Cellula staminale

• Cellula capace di dare origine a tutte le popolazioni cellulari di uno o più tessuti.

• Normalmente, da cellule staminali nascono sia nuove cellule staminali, sia cellule capaci di dividersi rapidamente, ma probabilmente solo un numero finito di volte, che cominciano anche il processo di differenziamento:

queste ultime costituiscono il cosiddetto compartimento di espansione del tessuto.

Stem cell niches

Direct contact Soluble factors Intermediate cell

stem cell

niche

NicheMicroenvironment around stem cells that provides support and signals regulating self-renewal and differentiation

Stem cell niches

The stem cell niche is a major concept in stem cell biology. Understanding the microenvironment around stem cells is as important as understanding stem cells themselves. The microenvironment regulates the behavior of stem cells and thus can teach us how to control stem cells in culture.

The niche can act on a stem cell by various mechanisms:•Direct contact between the stem cell and the niche cells•Soluble factors released by the niche that travel to the stem cell•Intermediate cells that ‘communicate’ between the niche and the stem cell

Scientists are still working to understand exactly how niches work, and more is known about the niches of some kinds of stem cells than others.

This selective gene expression controls the four essential processes bywhich the embryo is constructed:

• cell proliferation, producing many cells from one

• cell specialization, creating cells with different characteristics at differentpositions

• cell interactions, coordinating the behavior of one cell with that of itsneighbors

• cell movement, rearranging the cells to form structured tissues and organs

TotipotenteTotipotente

PluripotentePluripotente

MultipotenteMultipotente

UnipotenteUnipotente

Cellule Staminali Cellule Staminali PluripotentiPluripotenti indotte indotte iPSCiPSC

Cellule Staminali del cancroCellule Staminali del cancro

Cellula staminale totipotente

Cellula capace di dare origine a tutte le

popolazioni cellulari dell'organismo

Tali sono le cellule all'interno della morula o della blastocisti, cioè

di stadi precoci dello sviluppo embrionale (prima set timana di

sviluppo dell'uomo).

Le cellule embrionali staminali (in sigla ESda Embryonic Stem cell ), hanno come caratteristica principale l'elevata capacitàdi differenziarsi in qualsiasi altro tipo cellulare. Inoltre è possibile mantenerle in coltura per un lungo periodo di tempo.

neurons

grow under conditions B

Embryonic stem (ES) cells:Challenges

embryonic stem cells

skin

grow under conditions A

blood

grow under conditions C

liver

grow under conditions D

?

In tutti i mammiferi le prime fasi dello sviluppo embrionale a partire dallo zigote avvengono grazie

agli RNA messaggeri (mRNA) e alle proteine di origine materna presenti nel citoplasma dell'oocita.

Cellula staminale pluripotente

Cellula capace di dare origine a più

popolazioni cellulari, in generale a

tutte quelle di un tessuto

ad esempio, tutte quelle del midollo osseo, o tutte

quelle dell'epitelio mucoso intestinale

Cellula staminale del funicolo ombelicale:

Cellula staminale, presumibilmente pluripotente e capace di dare origine a varie popolazioni delle cellule del sangue, che è presente in circolo durante la vita fetale e quindi anche nel sangue del funicolo ombelicale; quest'ultimo può essere prelevato alla nascita senza alcun trauma per l'organismo.

Tissue stem cells:Neural stem cells (NSCs)

NSC

braincommitted progenitors specialized cells

Neurons

Interneurons

Oligodendrocytes

Type 2 Astrocytes

Type 1 Astrocytes

Cellule Staminali nell’adulto

muscles

skin

surface of the eye brain

breast

intestines (gut)

bone marrow

testicles

Nell'individuo adulto si trovano cellule staminali in diversi distretti tissutali

differenziati:

• nel midollo spinale

• nell'epitelio seminifero della gonade maschile

• nella retina

• negli epiteli

• nel cervello

Le cellule staminali adulte sono purtroppo di diffi cile

reperibilità, poiché numericamente molto scarse;

non possono essere coltivate a lungo poiché, dopo

alcune divisioni cellulari, tendono a perdere le

caratteristiche di pluripotenzialità.

Le cellule staminàli embrionali, invece, possono ess ere

mantenute in coltura per moltissimi cicli di divisi one,

addirittura per più di dieci anni, senza perdere di

pluripótenzialità.

Accanto a queste sorgenti fisiologiche di

cellule staminali, negli ultimi anni se ne è

aggiunta un'altra molto promettente, basata

sulla possibilità di modificare il programma

genetico delle cellule differenziate.

Induced pluripotent stem cells (iPS cells)

adult cell

‘genetic reprogramming’= add certain genes to the cell

induced pluripotent stem (iPS) cell

behaves like an embryonic stem cell

Advantage: no need for embryos! all possible types ofspecialized cells

culture iPS cells in the lab

differentiation

Induced pluripotent stem cells (iPS cells )

adult cell (skin)

genetic reprogramming

pluripotent stem cell(iPS)

differentiation

Geni della pluripotenza – Fattori di trascrizione

Nanog, Oct4, Sox2

Role of SOX2 in maintaining pluripotency of humanembryonic stem cells

Human embryonic stem cell (ESC) pluripotency is thought to be regulated byseveral keytranscription factors including OCT4, NANOG, and SOX2. Although the functions of OCT4and NANOG in human ESCs are welldefined, that of SOX2 has not been fullycharacterized. Toinvestigate the role of SOX2, wemodulated the level of SOX2 expression in human ESCs.Reduction of SOX2 expression in humanESCs induced trophectodermal and partialendodermaldifferentiation.

Genes to Cells (2010) 15, 455–469

Transcripts of NANOG and OCT4 have been recently identified in human t(4;11) leukemia and in a

model system expressing both t(4;11) fusion proteins. Moreover, downstream target genes of

NANOG/ OCT4/SOX2 were shown to be transcriptionally activated. However, the NANOG1 gene

belongs to a gene family, including a gene tandem duplication

(named NANOG2 or NANOGP1) and several pseudogenes (NANOGP2-P11). Thus, it was unclear

which of the NANOG family members were transcribed in t(4;11) leukemia cells. 50-RACE

experiments revealed novel 50-exons of NANOG1 and NANOG2, which could give rise to the

expression of two different NANOG1 and three different NANOG2 protein

variants. Moreover, a novel PCR-based method was established that allows distinguishing between

transcripts deriving from NANOG1, NANOG2 and all other NANOG pseudogenes (P2–P11). By

applying this method, we were able to demonstrate that human hematopoietic stem cells and

different leukemic cells transcribe NANOG2. Furthermore, we nctionally tested NANOG1 and NANOG2

protein variants by recombinant expression in 293 cells. These studies revealed that NANOG1 and

NANOG2 protein variants are functionally equivalent

and activate a regulatory circuit that activates specific stem cell genes. Therefore, we pose the

hypothesis that the transcriptional activation of NANOG2 represents a ‘gain-of-stem cell function’ in

acute leukemia.

Transcriptional properties of human NANOG1 and NANOG2 in acute leukemic cells5384–5395 Nucleic Acids Research, 2010, Vol. 38, No. 16 Published online 28 April 2010doi:10.1093/nar/gkq307

EMBO J. 2010 Oct 6;29(19):3236-48. Epub 2010 Aug 24.

Oct-3/4 regulates stem cell identity and cell fate decis ions by modulating Wnt/ β-catenin signalling.

Abu-Remaileh M, Gerson A, Farago M, Nathan G, Alkalay I, Zins Rousso S, Gur M, Fainsod A, Bergman Y.

Department of Developmental Biology and Cancer Research, The Hebrew UniversityMedical School, Jerusalem, Israel.

AbstractAlthough the transcriptional regulatory events triggered by Oct-3/4 are well documented,

understanding the proteomic networks that mediate the diverse functions of this POU domain homeobox protein remains a major challenge. Here, we present genetic and

biochemical studies that suggest an unexpected novel strategy for Oct-3/4-dependent regulation of embryogenesis and cell lineage determination. Our data suggest that Oct-3/4

specifically interacts with nuclear β-catenin and facilitates its proteasomal degradation, resulting in the maintenance of an undifferentiated, early embryonic phenotype both in Xenopus embryos and embryonic stem (ES) cells. Our data also show that Oct-3/4-mediated control of β-catenin stability has an important function in regulating ES cell

motility. Down-regulation of Oct-3/4 increases β-catenin protein levels, enhancing Wntsignalling and initiating invasive cellular activity characteristic of epithelial-mesenchymal

transition. Our data suggest a novel mode of regulation by which a delicate balancebetween β-catenin, Tcf3 and Oct-3/4 regulates maintenance of stem cell identity. Altering

the balance between these proteins can direct cell fate decisions and differentiation.

Models of cellular reprogramming.

Stadtfeld M , Hochedlinger K Genes Dev. 2010;24:223 9-2263

©2010 by Cold Spring Harbor Laboratory Press

Putative role of reprogramming factors during iPSC formation.

Stadtfeld M , Hochedlinger K Genes Dev. 2010;24:223 9-2263

©2010 by Cold Spring Harbor Laboratory Press

CloningThere are two VERY different types of cloning:

Reproductive cloning

Use to make two identical individuals

Very difficult to do

Illegal to do on humans

Molecular cloning

Use to study what a gene does

Routine in the biology labs

gene 1

gene 2

Reproductive cloning

egg

take the nucleus (containing DNA)

remove nucleus and take the

rest of the cell

adult cell

Cloneidentical to the individual

that gave the nucleus

Dolly the sheep

È chiaro che disporre di un reagente biologico quale le cellule staminali, da differenziare nei diversi tipi cellulari, apre nuovi scenari terapeutici:

patologie ora poco trattabili, potrebbero essere affrontate con maggior successo grazie alla sostituzione dei tessuti

danneggiati.

Potential applications of iPSCs.

Stadtfeld M , Hochedlinger K Genes Dev. 2010;24:223 9-2263

©2010 by Cold Spring Harbor Laboratory Press

Spinal muscular atrophy refers toa group of autosomal recessive neuromuscular disorderscharacterized by degeneration ofthe anterior horn cells of the spinal cord, leading tosymmetrical muscle weaknessand atrophy. SMA is the secondmost common lethal, autosomalrecessive disease in Caucasiansafter cystic fibrosis( Wirth, 2000).

spinal muscular atrophy type I (SMA I) is caused by mutation or deletion in the copy of the SMN gene, known as SMN1

• ricostruzione del midollo spinale danneggiato da traumi fisici

• del tessuto cardiaco dopo infarto, • malattie infiammatorie di natura sistemica • a quelle muscolo-scheletriche (displasia ossea,

malattie progressive delle giunture, osteogenesi imperfetta, miopatie primitive)

• malattie degenerative della retina, • della cornea • dell'apparato uditivo, • malattie degenerative del sistema nervoso

(Alzheimer, Parkinson, malattia di Huntington, sclerosi laterale amiotrofica)

Applicazioni terapeutiche delle cellule staminali

· Trapianto autologo di cellule staminali emopoietiche

· Trapianto allogenico di cellule staminali

· Trapianto allogenico di cellule staminali emopoietiche del cordone ombelicale

· Trapianto di cellule staminali cutanee: cellule staminali coltivate in vitro utilizzabili solo per pazienti con patologie cutanee gravi ed ustioni

Terapia genica

con le tecniche d'Ingegneria genetica si può

correggere l'effetto prodotto da geni difettosi infatti le

cellule staminali tollerano meglio di altre cellule

l'inserimento di geni dall'esterno.

Questa tecnica potrebbe permettere la correzione di

difetti genetici nelle prime fasi di sviluppo

embrionale

Rigenerazione di tessuti ed organi a partire da cellule staminali:

E' già possibile ricostituire alcune popolazioni cellulari a partire da cellule staminali pluripotenti del tessuto medesimo, come avviene per le cellule del sangue mediante il trapianto di midollo osseo;

sono allo studio progetti per far rigenerare interi tessuti o addirittura organi,

ma va tenuto presente che in questo caso non èsufficiente far generare una o più popolazioni cellulari, ma va anche fatto in modo che queste producano matrice extracellulare nella appropriata quantità e disposizione, che assumano i rapporti spaziali reciproci tipici del tessuto e dell'organo maturo e, in particolare per la rigenerazione di organi, che questi assumano la forma tridimensionale loro propria e necessaria alla funzione, tutti punti ancora da esplorare.

Cancer Stem Cells

cancer stem cells (csc)

• SELF-RENEWAL• DIFFERENTIATION• PROLIFERATIVE ABILITY

ABERRANT REGULATION

Modified from Bjerkvig R et al. Nat Rev Cancer. 2005;5:899-904

Minority of cancer cells with tumorigenic potential

NORMAL

TUMORAL

Cancer stem cell theory

New cancer model:1) Tumors arise from cells termed cancer stem cells that

have properties of normal stem cells, particularly self-renewal and multipotentiality (a minority) of tumor cells.

2) Unregulated cell growth is due to a disruption in the regulatory mechanism in stem cell renewal.

3) Cancer is a stem cell disorder and not a simple mechanism whereby cell proliferation is disrupted.

Cancer stem cell theory

• These CSCs cells persist in tumors as a distinct population that likely causes relapses and metastasis.

• This theory explains why are manycancers so difficult to treat.

Cancer stem cell theory

Why stem cells?

• Only stem cells have the ability to self renewand neoplasia is essentially dysregulated self renewal

• Stem cells are long-lived cells which can acquire the necessary number of sequential mutations to convert a normal cell into a malignant one.

Are we targeting the right cells?

• Conventional chemotherapies kill differentiated or differentiating cells, which form the bulk of the tumor but are unable to generate a new one.

• A population of CSCs, which gave rise to it, remains untouched and may cause a relapse of the disease.

• Development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease, where little progress has been made in recent years.

WRONG TARGET. Traditional cancer therapies (top) kill rapidly dividing tumor cells (blue) but may spare stem cells (yellow) that can give rise to a new tumor. In theory, killing cancer stem cells (bottom) should halt a tumor's growth lead to its disappearance.

“Abbiamo finalmente un nuovo eccezionale obiettivo per la ricerca: le

staminali del cancro”, ha dichiarato oggi il Professor Umberto Veronesi, Direttore

Scientifico dell’Istituto Europeo di Oncologia di Milano, nell’ambito dell’annuale

presentazione dei risultati e dei programmi di ricerca dello IEO. “Le cellule

staminali tumorali non sono altro che cellule, più indifferenziate delle altre, che

sono in grado di alimentare la proliferazione del tumore. Sono in pratica il

serbatoio dal quale il cancro si approvvigiona. Essere capaci di intervenire su

questo serbatoio potrebbe quindi rappresentare la svolta finale per la soluzione

globale del problema cancro.

Cancer stem cells models

• Acute myelogenous leukemia : [CD34+,CD38-]

• Breast Cancer : [CD44+, CD24-/low]

• Brain tumor : [CD133+]

• Prostate cancer : [CD44+]

• Colon cancer : [CD133+]

Metastatic Cancer Cells =

Migratory Cancer Stem Cells

There are many molecular links between the regulation of normal embryogenesis and oncogenesis.

A different, and obvious, parallel between embryogenesis and oncogenesis can be observed in the spontaneous formation of tumors in the gonads of mammals, including humans.

These unusual tumors, which include teratomas, embryonal carcinomas, and teratocarcinomas, develop from the germ cells in the testes or ovaries. The tumors have provoked a great deal of interest because they often contain highly differentiated cells and tissues such as teeth, hair, neural cells, and epithelial cells. The structures are disorganized, but often recognizable.

Although teratomas are benign, embryonal carcinomas and teratocarcinomas are highly malignant. The latter contain a kind of stem cell, called an embryonal carcinoma (EC) cell, which in mice and humans resembles embryonic stem (ES) cells.

Some genes recently identified as important in the development of human cancers are also active during embryonic development.

For instance, the human breast cancer genes, BRCA1 and BRCA2, and their counterparts in mice are expressed in the three primary germ layers during embryogenesis, particularly in cell types undergoing the most rapid proliferation.

The expression of these genes is dependent on the stage of the cell cycle, with peak expression during the G1/S transition and lowest expression in cells in the G1 or G0 phase. In humans, BRCA1 and BRCA2 probably function during the development of mammary epithelium, although little is known about their role in this process.

For instance, Oct-4 is a transcription factor that has come to be recognized as a prototypical marker of undifferentiated, dividing cells.

It is necessary for maintaining the undifferentiated state and proliferation of cells of the inner cell mass and the epiblast.

Oct-4 is expressed in the mouse oocyte, it disappears during the first cleavage of the zygote, and it reappears in the four-cell mouse embryo as the genome of the zygote begins to control embryonic development.

Oct-4 is a member of the class 5 POU (for Pit, Oct, and Unc) family of transcription factors, which bind promoter or enhancer sites in DNA.

The transcription factor Oct-4 can activate or repress gene expression.

A target of Oct-4 in mouse embryogenesis is the Fgf4 gene. It encodes fibroblast growth factor-4 (FGF-4), a growth factor protein that is expressed together with Oct-4 in the inner cell mass and epiblast .FGF-4 is a paracrine signal, meaning that it is released from one cell type and it acts on another.

A series of recent experiments indicates that the level of Oct-4 expression—not simply its presence or absence in a cell—determines how mouse embryonic stem cells differentiate and whether they continue to proliferate