Cenni di istologia
• Ontogenesi
– Embriogenesi: precursori nel sacco
vitellino, strutture vascolari della regione
aorta gonade-mesonefro
– II mese: epatica che prosegue fino al VII
assieme alla splenica
– Al V mese mielopoiesi midollare: inizia
nelle ossa piatte e lunghe;
nell’invecchiamento residua nelle vertebre,
sterno, coste, ali iliache
CELLULE STAMINALI
Il potenziale multidifferenziativo della SC caratterizza due
principali tipi di SC: le SC ottenute da embrioni e le SC ottenute
da soggetto adulto.
Le SC embrionali si ottengono mediante la procedura del
trasferimento nucleare: il nucleo di una cellula somatica viene
inserito all’interno di un ovulo denucleato. Si sviluppa una
blastocisti dalla quale possono essere isolate SC, che possono
essere espanse in coltura e, indotte a differenziare verso un
determinato tipo di tessuto. Si tratta verosimilmente di SC
totipotenti.
Obiezioni etiche e problemi tecnici rendono assai problematica
la loro applicazione.
Le SC adulte non sollevano problemi di natura etica. Sono
facilmente ottenibili, posseggono una minor capacità
espansiva “in vitro” rispetto alle SC embrionali, sono
multipotenti
(ma
non
totipotenti),
hanno
capacità
differenziativa limitata a specifici tessuti, sono facilmente
applicabili dal punto di vista clinico.
Caratteristiche
• Capacità di bilanciare
autorinnovamento vs differenziazione
• Sono multipotenti fino a 10 distinte
linee cellulari
• Robusto potenziale proliferativo
• Rare 1:100.000
• Sono quiescenti o in ciclo lento
Dogma dell’embriologia
• Presenti solo in tessuti capaci di
rinnovamento e riparazione
• Dipendenza dal tipo di tessuto
determinato durante lo sviluppo fetale
• In ogni caso è rispettata l’origine dal
foglietto germinativo embrionale
Cellule staminali che rispettano
il dogma
• Staminali epatiche
• Staminali corneali
• Ematopoietiche
– Mieloidi
– Eritroidi
– Megacariocitiche
– Linfatiche
Differenziazione
delle cellule
emopoietiche
Cellule Staminali Ematopoietiche
• Mantenimento dell’emopoiesi per tutta
la vita
• Allotrapianto (familiare, MUD, aploidentico, singenico)
• Autotrapianto
• Mobilizzazione
• Purging
• Tests in vitro
• Tests in vivo (topi nudi, capre fetali)
CFU-GEMM
Mielopoiesi:CS multipotente formante colonie miste (granulo-eritromacrofagico-megacariocitarie)
CFU-GM
Mielopoiesi : Cellula formante colonie granulo-monocitiche
CFU-E
Eritropoiesi: cellula che forma piccoli aggregati eritroidi, la BFU-E grandi
colonie eritroidi
PLASTICITA’ delle cellule STAMINALI
• Studi in vivo ed vitro negli ultimi anni hanno evidenziato che in
individui adulti il potenziale delle cellule staminali NON sia
ristretto irreversibilmente dalla permanenza in un determinato
organo.
• In modelli animali cellule staminali del SNC o del muscolo
possono generare cellule emopoietiche, mentre c. midollari
possono generare c.muscolari, nervose endoteliali, epatiche.
• Meccanismo di azione : de-differenziazione? Transdifferenziazione? Fusione cellulare ?
• Meccanismi di regolazione: fattori solubili? Contatto diretto
cellula-cellula?
• Le cellule staminali nei tessuti adulti hanno capacità proliferativa
e differenziativa enorme simili alle c.staminali embrionali che
sono all’interno della blastocisti nella fase di pre-impianto
nell’endometrio.
Muscle regeneration by bone marrow derived myogenic progenitors
Ferrari G et Al Science 1998
Regeneration of ischemic cardiac muscle and vascular
endothelium by adult stem cells
Kathyjo A. Jackson
Incorporation of SP cells into
cardiomyocytes. (a) Negative control:
C57Bl/6 cardiac tissue stained for lacZ
expression. (b) Positive control: C57Bl/6Rosa26 cardiac tissue stained for lacZ
expression. This typical section
demonstrates both patterns of punctate
and whole-fiber staining. (c) Cross-section
of a heart from an SP cell–transplant
recipient, which received an infarct. (d)
Longitudinal section of an SP cell–
transplant recipient, which received an
infarct. (e–h) LacZ and -actinin costaining
of lacZ-positive fibers. (i) CD45 costaining
of the section in g and h. (j) Anti-CD45
staining of spleen (positive control).
Sections were stained with X-gal, and
LacZ-positive sections were subsequently
stained for -actinin (f, h) and CD45 (i),
and the sections were photographed.
Regeneration of ischemic cardiac muscle and vascular
endothelium by adult stem cells
Kathyjo A. Jackson
LacZ staining occurs primarily at the border of
myocardial infarction. (a) Lower-power (x10)
photograph of mouse myocardial infarction
after 4 weeks. The arrowhead points to the
location of lacZ staining shown in b and c. The
lighter pink tissue to the left and above the
arrowhead is primarily fibrotic and results from
the infarction. (b) Higher-power (x20)
photograph of the same section dual stained
for lacZ and the antimacrophage Ab F480. The
open arrowhead indicates a macrophage, the
closed arrowhead indicates lacZ-positive
cardiomyocytes (the same region shown in
Figure 4, g and h). (c) Higher-power
photograph of the same section (x40). (d)
Macrophage density of a cardiac section after 1
hour of ischemia and 3 hours of reperfusion.
The open arrowheads indicate two of the many
macrophages present. The counterstain is
eosin.
NEO-RIVASCOLARIZZAZIONE DI ZONE
ISCHEMICHE. Kawamoto A et al, 2003
a, Representative findings of NOGA
electromechanical mapping before (top) and 4
weeks after (bottom) NA/CD31+ MNC
transplantation. Brown dots in pretreatment map
show sites of cell transplantation. Red area on
pretreatment linear local shortening map (top
right) indicates area of decreased wall motion in
lateral wall of left ventricle, consistent with
ischemia in territory of LCx. Four weeks after local
CD31+ cell transplantation, this area of ischemia is
no longer evident (bottom right). b, Representative
findings of NOGA electromechanical mapping
before and 4 weeks after NA/CD31- MNCs
transplantation. Area of ischemia on pretreatment
map (top right) is unchanged or slightly increased
4 weeks after local transplantation of CD31- cells.
c, Representative findings of NOGA
electromechanical mapping before and 4 weeks
after PBS injection reveal findings similar to those
in CD31- transplant animals, with no improvement
in ischemic area. d, Change in percentage
ischemic area during 4 weeks after treatment.
NA/CD31+, swine receiving NA/CD31+ MNCs;
NA/CD31-, swine receiving NA/CD31- MNCs.
*P<0.05; **P<0.01.
Influence of mobilized stem cells on myocardial
infarct repair in a nonhuman primate model
Franc¸oise Norol, et Al. Blood 102: 4361; 2003
Generalized potential of adult neural stem cell
Clarke DL et Al Science 2000
Turning Brain into Blood: A Hematopoietic Fate
Adopted by
Adult Neural Stem Cells in Vivo
Christopher R. R. Bjornson,*†‡ Rodney L. Rietze,*§Brent A. Reynolds, M. Cristina Magli, Angelo
L. Vescovi Science 1999, 283:
Turning Brain into Blood: A Hematopoietic Fate
Adopted by
Adult Neural Stem Cells in Vivo
NSCs produce early hematopoieticcells after
transplantationinto irradiated Balb/c recipients.
Turning blood into brain Mezey E et al 2000
Y chromosome staining in the CNS. Coronal sections from 4month-old nontransplanted (A) female and (B) male brains were
mounted and processed together. The panels show the overlay
of the NeuN (red) immunostaining, Y chromosome
nonradioactive ISH [visualized with tyramide-FITC conjugate
(green)], and DAPI staining of cell nuclei (blue). The Y
chromosome was restricted to the male brain, demonstrating
hybridization specificity. (C) Confocal image of coronal sections
from a 4-month-old recipient female striatum that was doubleimmunostained for the neuron-specific antigens NeuN and NSE.
All NeuN-expressing cells (red) were also immunoreactive for
NSE (green). (D) Sagittal section from a 1-month-old female
PU.1 knockout mouse brain transplanted at birth with male bone
marrow. The Y chromosome was visualized with BCIP/NBT
(dark purple dots) to identify anatomical landmarks. cc, corpus
callosum; cx, cerebral cortex; CPu, caudate putamen; fi, fimbria
hippocampi; hi, hippocampus; LV, lateral ventricle. (E to G)
Identical fields showing NeuN, Y chromosome, and DAPI
nuclear triple staining in the hypothalamic dorsomedial nucleus
of a 3-month-old female recipient. Colocalization of the Y
chromosome [visualized with tyramide-FITC conjugate (green)]
to a NeuN immunopositive (red) nucleus is shown in (E). In (F),
DAPI staining identifies all cell nuclei (blue). Overlays of the
NeuN, Y chromosome, and DAPI fluorescence are shown in (G).
The arrow identifies a cell nucleus that contained both the Y
chromosome (indicating the bone marrow origin) and NeuN.
Scale bar in (G) represents the following sizes: 30 µm, (A) and
(B); 10 µm, (C); 250 µm (D); and 12 µm, (E) to (G). Similar
results were observed with three different animals for each
experimental condition
Cellule mesenchimali umane
ALTRE CELLULE STAMINALI MIDOLLARI
Nel midollo emopoietico sono presenti cellule staminali mesenchimali
(CSM), le quali sono capaci di dare origine ad osteoblasti, condroblasti
adipociti, cellule di tessuto muscolare liscio e scheletrico
Differenziazione in osteoblasti
IPOTESI