Prof.Dr.Dan Colita

Institutul Clinic Fundeni, Bucuresti

Oradea, 2012

STEM CELLS (SC)

SC = Four vital capacities

To divide for infinite periods

To self renew

To generate differentiated and highly specialised daughter cells

To persist throughout the life

Tissue or organ specific

functional cells, terminally

differentiated

Differentiation

Maturation

Self-renewal

The protagonists of the development are the

stem cells

zigot

Blastocyst

Inner cell mass

Embryo

The founder cell Embryonic SC

Totipotent Pluripotent Multipotent

Organogenesis (the fetus)

The post natal life

Adult somatic SC

The totipotent SC: the fertilised

egg

Unrestricted differentiation potential

Can give rise to all cells necessary for the

development of foetal and adult organs

The pluripotent SC = the

embryonic cells

Derived from the inner cell mass of the blastocyst at 4-5 days after the fertilization

Give rise to a variety of specialised cell types but unable to support the development of a foetus

Selected and cultured in vitro they can form spontaneous colonies (the”embryonic bodies”), which contain the precursor elements for many cell types of the embryonic and extraembryonic tissues (the pluripotency)

In appropriate ex vivo conditions they can generate differentiated offsprings, e.g. neuronal, hematopoietic, endothelial, contracting cardiomyocytes, insulin producing cells and others

Pera M.F., ASH 2002, the Education Program Book, pag 374

The hematopoietic SC in the

adult life

On the way of their differentiation they change from multipotent to bi-and monopotent SC and give rise finally to the eleven different mature myeloid and lymphoid cell species that populate the blood

Their main residence is the bone marrow but they have the capacity to mobilise in the circulation and homing afterwards in other seats (bone marrow and other tissues or organs)

They have a definite phenotypic profile (CD34+, CD133+, CD45+, c-kit +, CD38-, Lin -)which help to recognise and isolate them for medical purposes

By transplantation they repopulate the aplastic BM and sustain the hematopoiesis lifelong

The bone marrow houses many

types of ADULT SC

Hematopoietic SC (HSC)

Mesenchimal SC (MSC)

Multipotent adult progenitor cells (MAPC)

Endothelial SC (ESC)

The “ side population” (SP)

The Mesenchimal SC (Stromal

cell precursors?) Present in the fetal and adult BM (as a primary reservoir) and also in

other tissues, with a frequency that declnes with age, as a population mitotically quiescent and longevive

Are multipotent giving rise to specific tissue cell with mesodermic origin (bone, cartilage, adipocytes, cardiomyocytes, bone marrow stroma) or non-mesodermic origin (neurons, astrocytes, oligodendrocytes, hepatocytes, epithelial cells)

They can be isolated in vitro (by their adherence on plastic dishes) and cultured

In cultures they secrete cytokines that support as”feeder layers” the proliferation and differentiation of HCS.

After in vivo transplantation they are capable of homing to the BM, sustain the engraftment and the hematopoietic reconstitution and to play a role in modulation of immune responses (by down regulating T- cell responses, they prevent or muzzle the GvH reaction)

The malignant SCs Cancers (Cc) are stem cell diseases

CC develops from a small subset of self renewal cells analogous to the organ SCs (‘tumor initiating cells or tumour stem cells)

Tu S.C Are rare

Generally not cycle active

Acquire the “malignant phenotype” after succesive genetic and epigenetic changes provoked by oncogenic hits

Engender rapidly dividing but terminally differentiated daughter cells that constitute the bulk of the tumor

Conventional chemotherapy – based tretament is primarily directed against the bulk of proliferating malignant cells and, thus, does not eliminate the quiescent tu S.C. (which constitute the origins of cancer recurrence and are responsible for the relapses) In the intention of cure, tu. S.C. must become the main targets of the

therapy E.g. the reaction of graft vs malignancy accompanying the HSC allotransplants for some

malignant hemopathy

Synergic action of antracycline + proteazome inhibitors against the AML stem cells in vitro

Clarke M.F & col, Cancer. Res, 2006, 66, 19

M.O. Hematopoieza (functia de salvare) Sange Pierderi naturale zilnice

maturatie

amplificare

diferentiere

recrutare

4 procese biologice 24 x 10 13 celule mature

9 specii

celulare

Progenitori

CSH

Precursori

Identificabili ai

seriilor

Fenotipare

Colonii

Mielograma Hemograma

3 functii vitale

transport

O2, CO2

aparare hemostaza

Er Gr (N,E,B)

MO

Lf (B.T)

NK

Tr

~24 x 10 11 celule

•2 x 10 11 Er

•10 11 Gr

•10 11 Tr

•10 11 Ly

TCSH

Stromal cells

VCAM1 SDF

CD34+

VLA 4 CXCR4

C - kit

The BM niche

CFA +G-CSF

G-CSF

AMD3100

CD34+

CD34+

CD34+

Release

Proliferation

Differentiation

Egress

OCL

Neu

IL8

MMP9

SDF 1

Untie

(Shedding)

Activation

Lapidot T, Petit I Exp hematolog 2002,30,973

Papayannopoulou T, Bood,2004, 103,1580

Liles NC & col, Blood,2002,100 (suppl,109 a)

Himerismul Coexistenta hematopoiezei gazdei cu aceea derivata din grefa

Depistare

• prin metoda FISH (fluorescent in situ hybridization) care depisteaza markerii cromozomilor X si Y (este aplicabila numai in cazurile in care donorii si recipientii sunt de sexe diferite)

• prin PCR (polymerase chain reaction) care analizeaza unele diferente dintre nucleotidele donorului si receptorului (STR = short tandem repeats)

• specificitate 1%

• sensibilitate (necesita 1-2 ng/mL de ADN

Exista adesea o necorelare intre chimerismul mieloid (care poate fi complet derivata din hematopoieza donorului) si chimerismul limfoid (care poate arata un chimerism mixt, de regula < 50%)

Studiul himerismului

• markerul grefarii reusite (chimerism 100% “de donor”). Himerismul complet semnifica eficienta grefei vs.limfohematopoieza receptorului si concomitent imunosupresia LfT. ale gazdei rezultata din intensitatea conditionarii care previne rejetul

• MAC vs. RIC Him.complet se atinge ~ in z. +80 !

• pierderea/neatingerea him.complet rejetul grefei

In conditiile RIC un chimerism < 50% in z. +14 al LfT., NK se asociaza cu un risc > 25% de rejectie

DLI conversie la himerism 100% in cca 75% dintre cazuri

cu scaderea ratei rejectiei la 25%

Chimerism with STR-PCR National Forensic Institute Bucharest

patient donor patient day+104

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8

D8S1179

T

-

Stable complete chimera

100

81.83 84.84

81.26

20.24 17.05 18.15

13.98 8.89

100

5.79 5.05 0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10 11 12

% D

on

or

% Donor

184 174 150= RT 143 134 127 119 106 70 57 42 T

12 11 10 9 8 7 6 5 4 3 2 1

Days post-Tx‏

T T

Progressive mixed chimera with rejection and re-transplantation

Grefa de CSH

Recoltata din M.O. direct (prin punctii osoase)

indirect (din sg.periferic, prin afereza)

Recoltata din cordon ombilical

Evolutia grefei dupa administrare

Grefare = reconstituirea functionala a hematopoiezei cu celule dezvoltate din

CSH administrate

= aprecierea grefarii a) ptr.neutrofile : nr.absolut > 500/L, trei zile la rand

b) ptr.plachete : > 20000/ L (in absenta transfuziilor de Tr.)

c) ptr.eritrocite : reticulocite > 30 x 106 sau > 1% (in absenta transfuziilor

de Er.)

Esecul grefarii a) primar : neatingerea pragului de > 500 neutrofile /L in ≥ 28 de zile

b) secundar : scaderea neutrofilelor sub prag, dupa grefare, necorectabila

cu factori de crestere, in absenta unei infectii sau al efectului medicatiei

Reconstituirea imuna post

Tx-1 Tx.CSH imunodeficienta profunda care se repara in luni - ani

predispozitie la evenimente amenintatoare de viata :

• infectii oportunistice bacteriene

• reactivarea unor infectii latente virale si micotice

• boli maligne

Reconstituirea imuna inseamna refacerea functionala a imunitatii innascute si a

imunitatii adaptative

• Imunitatea innascuta (nespecifica, rapida)

Efectori : celule efectoare derivate din progenitori mieloizi : NK,

cel.dendritice, Mcf./M, PMN

• Imunitatea adaptativa (specifica)

Efectori : celule efectoare derivate din progenitori limfoizi : LfB

LfT : TH(CD4+)

Tcit(CD8+)

Treg Raspuns mediat de receptori specifici de Atg. ce implica

expansiunea clonala ce sta la baza amplitudinii raspunsului si

formarea unor LfB si T longevive (celule de memorie) care persista

in stare de repaos pana la reaintalnirea cu Atg. specific cand produc

raspunsul secundar

Reconstituirea imuna post

Tx-2

Celule efectoare Fenotip (CD-uri)

LfB 19+, 20+

LfT 3+, 4+, 8+

CD4+ naïve 4+, 45RA+

CD4+ de memorie 4+, 45RO+

CD8+ naïve 8+, 45RA+,62L+, 11a+, 27+, 28+

CD8+ de memorie 8+, 45RO+, 45RA-, 27-

T reg 4+, 25+, Foxp3+

Celule dendritice HLA DR+, Lin-

Tiul I HLA DR+, Lin-, 11c+

Tipul II HLA DR+, Lin-, 123+

Celule NK 3-, 16+, 56+

Efectoare 3-, 16bright, 56dim

Reglatoare 3-, 16dim/-, 56bright

Monocite 14+, 15-

Markerii fenotipici ai celulelor imune efectoare comune

(dupa Kang Y & col, 2009)

Reconstituirea imuna post

Tx-3 Regenerarea celulara = recapituleaza ontogeneza normala

1. Regenerarea LfB

Surse : CS si LfB ale donorului care au supravietuit conditionarii

CS si LfB ale donorului transferate cu grefa

Fenotipurile precursorilor B sunt CD10+ si CD19+, insotite ulterior de alti Atg specifici liniei B

In primele 3 luni post Tx sunt reduse nr.LfB circulante, nivelele Ig serice si raspunsurile LfB

la stimulare antigenica

Normalizarea titrurilor Ig M : 6 luni

G : 12 luni

A : 24 luni

~ Z + 90 se recapata capacitatea de realizare a afinitatii depline a Atc. la Atg. (prin refacerea

capacitatilor de mutatii somatice ale genei VH)

2. Regenerarea LfT

Surse : progenitori T si LfT mature ale receptorului care au supravietuit conditionarii

LfT mature cu donor transferate cu grefa

LfT precursoare diferentiate din grefa

CS ale grefei

Doua stadii : 1. timpuriu : LfT mature ale donorului transferate cu grefa primele celule

efectoare T (au un repertoriu ingustat de reactii si pot persista

10-20 ani)

2. tardiv : LfT naive diferentiate din CS grefate la nivelul timusului (au un

repertoriu mai diversificat de reactii). Productia intratimica este

dependenta de provenienta donatorului (din familie sau nu) si de

absenta rGvH cronica

Reconstituirea imuna post

Tx-4 3. Reconstituirea celulelor NK

Surse : aceiasi precursori ca celulele T

Maturatie in M.O., nu necesita prezenta unui timus functional

Sunt primele celule care se refac dupa Tx (primele 2-3 sapt.)

Rol in efectul GvM

Ucid celulele tinta care nu exprima Atg HLA cls.I ale selfului

4. Reconstituirea celulelor prezentatoare de Atg.

Celulele dendritice (prezentatoare de Atg) provin din monocitele circulante,

care, la randul lor, deriva din CS medulare

Exista 2 tipuri de CD :

CD I

Lin-, HLA.DR+, 11c+

• promoveaza raspunsurile imune tip TH 1

productia citokinelor proinflamatorii

TNF

IL12

IFN

CD II Lin-, HLA.DR+, 123+

• promoveaza raspunsurile tip TH2

productia citokinelor antiinflamatorii

IL4

IL10

• deprima productia

TNF

IFN

• afecteaza efectul GvM

Sindromul de grefare

Criterii majore (M)

t◦ ≥ 38,3 C neinfectioasa

rash > 25% din s.c., nelegat de medicatie

infiltrate pulmonare, EPA si SaO2 < 90%

Criterii minore (m)

BRT ≥ 2mg/dL sau transaminazemie > 2 x normalul

creatinina > 2 x normalul

crestere ponderala > 2,25 x nivelul de baza

encefalopatie tranzitorie “sine materia”

Dgn. 3 M sau 2M + 1 m (minimum)

Frecventa : 10 – 50% cazuri post TCSH

Cauze probabile : efecte toxice ale conditionarii, producte rezultate din

degranularea neutrofilelor , efectul unor mediatori de inflamatie (IL1, TNF,

IFN) eliberati din celulele endoteliale sau din Lf.T.

Tratament : CS 1 mg/kg/zi, i.v. ameliorare in 3-5 zile

Accident acut, cu potential fatal ce preceda grefarea neutrofilelor cu cateva zile

To be remember

SC are the sine qua non condition for the existence

The homeostatic maintenance of most tissues capable of regeneration and repair is ultimately mediated by tissue-specific stem cells

HSC are the first tissue-specific stem cells to be prospectively isolated and are, to date, the only stem cells in routine clinical use

The plasticity phenomenon place the stem cell biology in the forefront of the researches for the regenerative medicine

The better knowledge of the properties of the malignant stem cells open new horisons in the treatment strategy of cancer

Stem cells ?

A Dilema !