CELLULE STAMINALICELLULE STAMINALIAPPLICAZIONI APPLICAZIONI

TERAPEUTICHETERAPEUTICHE

Maurilio SampaolesiMaurilio SampaolesiMaurilio SampaolesiMaurilio Sampaolesi

LESSON 7LESSON 7Corso ANATOMIA UMANA IICorso ANATOMIA UMANA IISpecialisticaSpecialistica(2007(2007--2008)2008)

APPARATO APPARATO TEGUMENTARIOTEGUMENTARIO

È COSTITUITO DALLA CUTE E DA UNO È COSTITUITO DALLA CUTE E DA UNO STRATO SOTTOCUTANEO COMPOSTO DA STRATO SOTTOCUTANEO COMPOSTO DA CONNETTIVO LASSO, E ANNESSI CUTANEICONNETTIVO LASSO, E ANNESSI CUTANEI

EPIDERMIDE (5 strati cellulari)EPIDERMIDE (5 strati cellulari)

1) Germinativo1) Germinativocellule basali (strato unico attaccato alla cellule basali (strato unico attaccato alla

lamina basale), 80% mitosi delle lamina basale), 80% mitosi delle epidermideepidermide

2) Spinoso 2) Spinoso cellule spinali (stratificato, cellule spinali (stratificato, desmosomi)desmosomi)

3) Granuloso3) Granuloso

••0, 30, 3--1,5 mm1,5 mm

CCHHEERRAATT

stem

3) Granuloso3) Granulosocellule produttrici di cheratoialina,cellule produttrici di cheratoialina,mitosi assentemitosi assente

4) Lucido4) LucidoCellule appiattite indistinguibile il Cellule appiattite indistinguibile il nucleo produttrici di eleidinanucleo produttrici di eleidina

5) Corneo5) Corneospessore variabile, molte cellule, le più spessore variabile, molte cellule, le più superficiali producono squamette superficiali producono squamette Desmosomi modificatiDesmosomi modificati

TTIINNOOCCIITTII

diff

•• Cellule immerse in una abbondante matriceCellule immerse in una abbondante matrice

fondamentali funzione:fondamentali funzione:

RIEMPIMENTORIEMPIMENTO e e RELAZIONARERELAZIONARE

gli altri tessuti, formando un’impalcatura gli altri tessuti, formando un’impalcatura

(stroma)(stroma)

CLASSIFICAZIONECLASSIFICAZIONE

•• Cellule immerse in una abbondante matriceCellule immerse in una abbondante matrice

fondamentali funzione:fondamentali funzione:

RIEMPIMENTORIEMPIMENTO e e RELAZIONARERELAZIONARE

gli altri tessuti, formando un’impalcatura gli altri tessuti, formando un’impalcatura

(stroma)(stroma)

CLASSIFICAZIONECLASSIFICAZIONE

TESSUTO CONNETTIVOTESSUTO CONNETTIVO

Tessuto connettivo propriamente detto Tessuto connettivo propriamente detto

Tessuto connettivo speciale Tessuto connettivo speciale

Tessuto connettivo propriamente detto Tessuto connettivo propriamente detto

Tessuto connettivo speciale Tessuto connettivo speciale

((adiposo,adiposo, cartilagineocartilagineo, , e e sanguesangue))osseoosseo

TESSUTO CONNETTIVOTESSUTO CONNETTIVO

•• La cuteLa cute è costituita da un è costituita da un epitelio pluristratificato,epitelio pluristratificato,l’l’epidermide epidermide e da uno strato e da uno strato di connettivo lasso, il di connettivo lasso, il dermaderma..

I I melanocitimelanociti sono localizzati sono localizzati nel derma e nello strato basalenel derma e nello strato basaledell’epidermidedell’epidermide

APPARATO APPARATO TEGUMENTARIOTEGUMENTARIO

dell’epidermidedell’epidermide

•• Il sottocutaneoIl sottocutaneo è costituito da un è costituito da un Tessuto connettivo lasso ed è percorsoTessuto connettivo lasso ed è percorsoda da vasi sanguignivasi sanguigni e e linfaticilinfatici, e , e fibre nervosefibre nervose(presenti anche nell’epidermide)(presenti anche nell’epidermide)

•• Gli annessi cutaneiGli annessi cutanei sono sono specializzazioni del derma e specializzazioni del derma e dell’epidermide divisi in cornei (peli e dell’epidermide divisi in cornei (peli e unghie) e ghiandolari (g.sebacee, unghie) e ghiandolari (g.sebacee, g.sudoripare e le mammelle)g.sudoripare e le mammelle)

0,70,7--1,4 mm1,4 mm

CUTE: epidermide, derma CUTE: epidermide, derma •• spessore 0,3spessore 0,3--4 mm4 mm•• Caratteristiche: colore, Caratteristiche: colore,

l’eleasticità, mobilitàl’eleasticità, mobilità•• Caratt.morfologiche: Caratt.morfologiche: --DepressioniDepressioni--DepressioniDepressioni(pieghe articolari, muscolari etc): (pieghe articolari, muscolari etc):

rughe, fossette e solchirughe, fossette e solchi--RilieviRilievipermanentipermanenti(polpastrelli, il rafe, creste cutanee)(polpastrelli, il rafe, creste cutanee)TransitoriTransitori(pelle arsina, (pelle arsina,

EPIDERMIDE (5strati cellulari)EPIDERMIDE (5strati cellulari)

Granuloso, LucidoGranuloso, LucidoAssenti nel resto del corpoAssenti nel resto del corpo

CorneoCorneoSpessore molto inferiore nel resto del Spessore molto inferiore nel resto del Spessore molto inferiore nel resto del Spessore molto inferiore nel resto del Corpo; forforaCorpo; forfora

DERMADERMAstrato subepitelialestrato subepiteliale

strato reticolarestrato reticolare

•• 0,20,2--3 mm3 mm•• PapillePapille•• Connetivo compattoConnetivo compatto•• Connetivo compattoConnetivo compatto•• MelanocitiMelanociti•• Miociti lisciMiociti lisci(muscoli erettori dei peli)(muscoli erettori dei peli)

Epidermal (and other lining epithelial) stem and transient Epidermal (and other lining epithelial) stem and transient amplifying cells can be identified in culture amplifying cells can be identified in culture (1, 2, 6(1, 2, 6--13)13). Indeed, . Indeed,

using clonal analysis, three types of keratinocytes with using clonal analysis, three types of keratinocytes with different capacities for multiplication have been characterized different capacities for multiplication have been characterized (6)(6). The holoclone, which is generated by the epidermal stem . The holoclone, which is generated by the epidermal stem cell cell (2, 6, 12, 13)(2, 6, 12, 13), has a tremendous potential for proliferative , has a tremendous potential for proliferative selfself--renewal, being able to undergo more than 140 doublings renewal, being able to undergo more than 140 doublings selfself--renewal, being able to undergo more than 140 doublings renewal, being able to undergo more than 140 doublings before senescence. The paraclone, which is generated by a before senescence. The paraclone, which is generated by a transient amplifying cell, has a very limited growth potential transient amplifying cell, has a very limited growth potential (being committed to a maximum of 15 divisions) and usually (being committed to a maximum of 15 divisions) and usually

gives rise to aborted colonies gives rise to aborted colonies (6)(6). The meroclone is an . The meroclone is an intermediate type of cell and is a reservoir of transient intermediate type of cell and is a reservoir of transient amplifying cells amplifying cells (2, 6)(2, 6). The transition from holoclone to . The transition from holoclone to meroclone to paraclone is an irreversible unidirectional meroclone to paraclone is an irreversible unidirectional

process that occurs slowly during ageing process that occurs slowly during ageing (6)(6)..

AbstractAbstract..Cell therapy is an emerging therapeutic strategy aimed at replacing or Cell therapy is an emerging therapeutic strategy aimed at replacing or repairing severely damaged tissues with cultured cells. Epidermal repairing severely damaged tissues with cultured cells. Epidermal

The control of epidermal stem cells (holoclones) in the The control of epidermal stem cells (holoclones) in the treatment of massive fulltreatment of massive full--thickness burns with autologous thickness burns with autologous

keratinocytes cultured on fibrinkeratinocytes cultured on fibrinPellegrini GPellegrini G, , Ranno RRanno R, , Stracuzzi GStracuzzi G, , Bondanza SBondanza S, , Guerra LGuerra L, , Zambruno GZambruno G, , Micali GMicali G, , De Luca MDe Luca MLaboratory of Tissue Engineering, I.D.I., Istituto Dermopatico dell'Immacolata, Rome, Italy.Laboratory of Tissue Engineering, I.D.I., Istituto Dermopatico dell'Immacolata, Rome, Italy.

Transplantation. 1999 Sep 27;68(6):868Transplantation. 1999 Sep 27;68(6):868--7979

repairing severely damaged tissues with cultured cells. Epidermal repairing severely damaged tissues with cultured cells. Epidermal regeneration obtained with autologous cultured keratinocytes (cultured regeneration obtained with autologous cultured keratinocytes (cultured autografts) can be lifeautografts) can be life--saving for patients suffering from massive fullsaving for patients suffering from massive full--thickness burns. However, the widespread use of cultured autografts has thickness burns. However, the widespread use of cultured autografts has been hampered by poor clinical results that have been consistently been hampered by poor clinical results that have been consistently reported by different burn units, even when cells were applied on properly reported by different burn units, even when cells were applied on properly prepared wound beds. This might arise from the depletion of epidermal prepared wound beds. This might arise from the depletion of epidermal stem cells (holoclones) in culture. Depletion of holoclones can occur stem cells (holoclones) in culture. Depletion of holoclones can occur because of (i) incorrect culture conditions, (ii) environmental damage of because of (i) incorrect culture conditions, (ii) environmental damage of the exposed basal layer of cultured grafts, or (iii) use of new substrates or the exposed basal layer of cultured grafts, or (iii) use of new substrates or culture technologies not pretested for holoclone preservation.culture technologies not pretested for holoclone preservation.

Figure Figure 1. Cultivation of keratinocytes on fibrin.1. Cultivation of keratinocytes on fibrin.(A) A sheet of fibrin sealant (prepared as (A) A sheet of fibrin sealant (prepared as described under described under Materials and MethodsMaterials and Methods) used ) used as a substrate for cultivation of secondary as a substrate for cultivation of secondary keratinocytes. (B) Growth kinetics of keratinocytes. (B) Growth kinetics of keratinocytes cultured on plastic or on fibrin. keratinocytes cultured on plastic or on fibrin. Subconfluent primary cultures (strain K71) were Subconfluent primary cultures (strain K71) were trypsinized and plated either on plastic (open trypsinized and plated either on plastic (open circles) or on fibrin (close circles) at a cell circles) or on fibrin (close circles) at a cell density of 6.5density of 6.5××110033 cells/cmcells/cm22. After 24 hr, . After 24 hr, unattached cells were removed. Epidermal unattached cells were removed. Epidermal growth factor was added at day 3. Cells were growth factor was added at day 3. Cells were trypsinized and counted every 24 hr in duplicate trypsinized and counted every 24 hr in duplicate until confluence was reached. Three different until confluence was reached. Three different cell strains were examined with similar results, cell strains were examined with similar results, cell strains were examined with similar results, cell strains were examined with similar results, and the average cell population doubling time and the average cell population doubling time was of approximately 21 hr in both conditions. was of approximately 21 hr in both conditions. Subconfluent primary cultures (strain K71) were Subconfluent primary cultures (strain K71) were plated either on plastic (C) or on fibrin (D) onto plated either on plastic (C) or on fibrin (D) onto lethally irradiated 3T3lethally irradiated 3T3--J2 cells (FL). KC indicate J2 cells (FL). KC indicate a keratinocyte colony after 6 days of cultivation. a keratinocyte colony after 6 days of cultivation. When such cultures where subconfluent (7 days When such cultures where subconfluent (7 days after plating), cells were trypsinized and CFE after plating), cells were trypsinized and CFE assays were performed by plating (in duplicate) assays were performed by plating (in duplicate) 500 cells onto lethally irradiated 3T3500 cells onto lethally irradiated 3T3--J2 cells. J2 cells. Colonies were stained with rhodamine B 14 Colonies were stained with rhodamine B 14 days later. (E) CFE from plasticdays later. (E) CFE from plastic--cultured cultured keratinocytes. (F) CFE from fibrinkeratinocytes. (F) CFE from fibrin--cultured cultured keratinocytes. Values of total/aborted colonies keratinocytes. Values of total/aborted colonies are 34.6%/3.4% in panel E and 39.7%/4.0% in are 34.6%/3.4% in panel E and 39.7%/4.0% in panel F.panel F.

CFE = efficiency colony formationCFE = efficiency colony formation

Figure 2. Figure 2. Clonal analysis. Single Clonal analysis. Single keratinocytes obtained from plastickeratinocytes obtained from plastic-- or or fibrinfibrin--cultured secondary keratinocytes cultured secondary keratinocytes (strain K49) were isolated under direct (strain K49) were isolated under direct vision and plated onto a regular feedervision and plated onto a regular feeder--layer in multiwell plates.layer in multiwell plates. After 7 days, After 7 days, clones were photographed and their areas clones were photographed and their areas were measured (as in A and B). Each clone were measured (as in A and B). Each clone was then transferred into two indicator was then transferred into two indicator dishes. One dish (3/4 of the clone) was dishes. One dish (3/4 of the clone) was used for serial propagation. The second used for serial propagation. The second dish (1/4 of the clone) was fixed 9dish (1/4 of the clone) was fixed 9--12 days 12 days later and stained with rhodamine B for later and stained with rhodamine B for classification of clonal type (as in C and classification of clonal type (as in C and D). Two holoclones (h) (see D). Two holoclones (h) (see ResultsResults) ) D). Two holoclones (h) (see D). Two holoclones (h) (see ResultsResults) ) obtained from plastic (A) or fibrin (B) obtained from plastic (A) or fibrin (B) cultures and their daughter colonies (C cultures and their daughter colonies (C and D, respectively) are shown here. and D, respectively) are shown here. Dishes in panels C and D were fixed and Dishes in panels C and D were fixed and stained 10 days after plating. (E) The stained 10 days after plating. (E) The number of holoclones, meroclones, and number of holoclones, meroclones, and paraclones isolated by clonal analysis of paraclones isolated by clonal analysis of keratinocyte strain K49. A total of 37 keratinocyte strain K49. A total of 37 plasticplastic--derived (green bars) and 39 fibrinderived (green bars) and 39 fibrin--derived (blue bars) clones were analyzed. derived (blue bars) clones were analyzed. Plastic: 6 holoclones, 19 meroclones, 12 Plastic: 6 holoclones, 19 meroclones, 12 paraclones. Fibrin: 8 holoclones, 23 paraclones. Fibrin: 8 holoclones, 23 meroclones, 8 paraclones.meroclones, 8 paraclones.

Table 1. Treatment of massive fullTable 1. Treatment of massive full--thickness burns with the composite thickness burns with the composite allodermis/fibrinallodermis/fibrin--cultured autograft (AD/Fcultured autograft (AD/F--CEA) techniquECEA) techniquE

Figure 3. Figure 3. Treatment of massive fullTreatment of massive full--thickness burns with the composite thickness burns with the composite AD/FAD/F--CEA technique.CEA technique. This patient This patient (K50) received full(K50) received full--thickness flame thickness flame burns on 45% of his body surface. burns on 45% of his body surface. Panel A shows his legs at admission. Panel A shows his legs at admission. Escharectomy was performed by Escharectomy was performed by tangential excision to viable tissue, tangential excision to viable tissue, and the receiving bed was and the receiving bed was completely covered by a 1:2 meshed completely covered by a 1:2 meshed donor skin (B). Panel C (at arrows) donor skin (B). Panel C (at arrows) shows the donor dermal network shows the donor dermal network remaining on the wound bed after remaining on the wound bed after remaining on the wound bed after remaining on the wound bed after removal of the allogenic epidermis, 2 removal of the allogenic epidermis, 2 weeks later. Panel D shows the weeks later. Panel D shows the donor dermal network covered with a donor dermal network covered with a fibrinfibrin--cultured autograft (at arrows). cultured autograft (at arrows). The take down (evaluated by visual The take down (evaluated by visual inspection 9 days after grafting) was inspection 9 days after grafting) was over 95% (E). At 1 year of followover 95% (E). At 1 year of follow--up, up, epidermal regeneration was epidermal regeneration was complete and stable, as shown in complete and stable, as shown in panels F and G.panels F and G.

allodermis/fibrinallodermis/fibrin--cultured autografts (AD/Fcultured autografts (AD/F--CEA) techniqueCEA) technique

Figure 4. Histology. Figure 4. Histology. Appearance of grafted Appearance of grafted epidermis at 4 (A, patient epidermis at 4 (A, patient K68), 8 (B, patient K60), K68), 8 (B, patient K60), and 12 (C, patient K50) and 12 (C, patient K50) months. Note the months. Note the months. Note the months. Note the progressive formation of progressive formation of rete ridges (semithin rete ridges (semithin sections,).sections,).

Figure 5. Figure 5. Electron microscopy. Electron Electron microscopy. Electron microscopy of grafted skin (Amicroscopy of grafted skin (A--E) and E) and normal control skin (F).normal control skin (F). (A) At 8 months (A) At 8 months after grafting (patient K60), a melanocyte after grafting (patient K60), a melanocyte (m) is visible between basal keratinocytes, (m) is visible between basal keratinocytes, which show cytoplasmic melanosomes which show cytoplasmic melanosomes (white asterisk); arrows denote the dermal(white asterisk); arrows denote the dermal--epidermal junction with keratinocyte epidermal junction with keratinocyte microfoot processes and continuous microfoot processes and continuous lamina densa. Numerous collagen fibers lamina densa. Numerous collagen fibers are arranged within the dermis (d) (are arranged within the dermis (d) (x4x4200). 200). (B) At 4 months after grafting (patient K68), (B) At 4 months after grafting (patient K68), a Langerhans cell (LC) is separated from a Langerhans cell (LC) is separated from the dermal epidermal junction (arrows) by the dermal epidermal junction (arrows) by the basal portion of a basal keratinocyte; the basal portion of a basal keratinocyte; note the mature melanosomes (asterisks) note the mature melanosomes (asterisks)

**

note the mature melanosomes (asterisks) note the mature melanosomes (asterisks) distributed within the cytoplasm of distributed within the cytoplasm of neighboring keratinocytes (neighboring keratinocytes (x5x5000); the 000); the inset shows a higher magnification of the inset shows a higher magnification of the Langerhans cell cytoplasm with typical Langerhans cell cytoplasm with typical Birbeck granules. Appearance of the Birbeck granules. Appearance of the dermaldermal--epidermal junction at 4 (C, patient epidermal junction at 4 (C, patient K68), 8 (D, patient K60), and 12 (E, patient K68), 8 (D, patient K60), and 12 (E, patient K50) months after grafting and in normal K50) months after grafting and in normal control skin (F): mature hemidesmosomes control skin (F): mature hemidesmosomes (asterisks) and a continuous basal lamina (asterisks) and a continuous basal lamina are visible in all specimens but in panel C are visible in all specimens but in panel C the basal lamina appears of normal the basal lamina appears of normal thickness only in correspondence to thickness only in correspondence to hemidesmosomes.hemidesmosomes.

RESULTSRESULTS: : We show that: (i) the relative percentage of holoclones, meroclones, and We show that: (i) the relative percentage of holoclones, meroclones, and paraclones is maintained when keratinocytes are cultivated on fibrin, paraclones is maintained when keratinocytes are cultivated on fibrin, proving that fibrin does not induce clonal conversion and consequent loss proving that fibrin does not induce clonal conversion and consequent loss of epidermal stem cells; (ii) the clonogenic ability, growth rate, and longof epidermal stem cells; (ii) the clonogenic ability, growth rate, and long--term proliferative potential are not affected by the new culture system; (iii) term proliferative potential are not affected by the new culture system; (iii) when fibrinwhen fibrin--cultured autografts bearing stem cells are applied on massive cultured autografts bearing stem cells are applied on massive fullfull--thickness burns, the "take" of keratinocytes is high, reproducible, and thickness burns, the "take" of keratinocytes is high, reproducible, and permanent; and (iv) fibrin allows a significant reduction of the cost of permanent; and (iv) fibrin allows a significant reduction of the cost of cultured autografts and eliminates problems related to their handling and cultured autografts and eliminates problems related to their handling and transportation. transportation. transportation. transportation.

CONCLUSIONCONCLUSION::Our data demonstrate that: (i) cultured autografts bearing stem cells can Our data demonstrate that: (i) cultured autografts bearing stem cells can indeed rapidly and permanently cover a large body surface; and (ii) fibrin is indeed rapidly and permanently cover a large body surface; and (ii) fibrin is a suitable substrate for keratinocyte cultivation and transplantation. These a suitable substrate for keratinocyte cultivation and transplantation. These data lend strength to the concept that the success of cell therapy at a data lend strength to the concept that the success of cell therapy at a clinical level requires cultivation and transplantation of stem cells. We clinical level requires cultivation and transplantation of stem cells. We therefore suggest that the proposal of a culture system aimed at the therefore suggest that the proposal of a culture system aimed at the replacement of any severely damaged selfreplacement of any severely damaged self--renewing tissue should be renewing tissue should be preceded by a careful evaluation of its stem cell population.preservation.preceded by a careful evaluation of its stem cell population.preservation.

APPARATO DELLA VISTAAPPARATO DELLA VISTAOCCHIO, APPARATO MOTOREOCCHIO, APPARATO MOTORE

E APPARATO PROTETTORE DELL’OCCHIOE APPARATO PROTETTORE DELL’OCCHIO

BULBO OCULARE DXBULBO OCULARE DX,,SEZ. ORIZZONTALESEZ. ORIZZONTALE

3 TONACHE:3 TONACHE:3 TONACHE:3 TONACHE:1)1) SCLEROTICASCLEROTICA(sclera e cornea)(sclera e cornea)

2)2) UVEAUVEA(corioidea, corpo (corioidea, corpo ciliare e l’iride)ciliare e l’iride)

3)3) RETINARETINA(visiva e cieca)(visiva e cieca)

membrana elasticamembrana elasticaesterna di Bowmannesterna di Bowmann

membrana elasticamembrana elasticainterna di Descemetinterna di Descemet

A, strati della CORNEAA, strati della CORNEAB, lamelle e stroma corneale B, lamelle e stroma corneale

parenchima corneale costituitoparenchima corneale costituitoda tessuto connetivo, in cui le da tessuto connetivo, in cui le

fibre collagene formano una serie fibre collagene formano una serie di circa 50 lamine (lamelle corneali) di circa 50 lamine (lamelle corneali)

La CORNEA è trasparente, non è vascolarizzata e si nutre grazie all’umorLa CORNEA è trasparente, non è vascolarizzata e si nutre grazie all’umoracqueo presente nella camera anteriore dell’occhio acqueo presente nella camera anteriore dell’occhio

T.A. cells = transient amplifying cellsT.A. cells = transient amplifying cells

p63p63

cornealcorneal

epidermalepidermal

PCNAPCNA

epidermalepidermal

Mutations in genes encoding the basement membrane

component laminin 5 (LAM5) cause junctional

epidermolysis bullosa (JEB)

published online 19 November 2006