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Le cellule staminali costituiscono la sorgente indifferenziata di tutti i possibili citotipi. Esse sono alla base delle varie fasi dello sviluppo, dai processi embriogenetici e morfogenetici alla crescita dell’organismo. Nell’adulto sono state rinvenute in pressocchè tutti i tessuti, non solo quelli di origine mesenchimale, compreso quello nervoso, e intervengono nel mantenimento di popolazioni cellulari sottoposte a turnover o nella rigenerazione. Dall’embrione allo stadio adulto la loro capacità differenziativa decresce progressivamente, passando dalla totipotenza tipica dei primissimi stadi della segmentazione, alla pluripotenza, vincolata al foglietto embrionale di origine, alla multipotenza, limitata ad alcune linee cellulari (addirittura una sola nel caso dell’unipotenza). Tuttavia è stato recentemente dimostrato che anche le cellule staminali adulte possiedono plasticità: il microambiente di residenza, infatti, guida il loro destino attraverso l’espressione di specifici fattori di crescita. Modificando artificialmente la composizione biochimica della nicchia staminale (per esempio variando il terreno di coltura in vitro o trapiantando le cellule in altri tessuti) è possibile indurre cellule multipotenti a transdifferenziare verso nuove linee. Questa scoperta ha rilanciato l’interesse nella medicina rigenerativa, precedentemente orientata maggiormente verso lo studio delle cellule embrionali, ma frenata da implicazioni etiche. Tecniche sempre più innovative mirano ad ampliarne le potenzialità e gli usi: dalla cura di malattie degenerative al trapianto autologo di tessuti ed organi (ingegneria tissutale), dal recupero funzionale in seguito a paralisi o traumi alle cure anti-invecchiamento, fino alla terapia antitumorale. Nuove prospettive nascono anche dall’individuazione di nuove fonti più facilmente accessibili di cellule staminali pluripotenti, come il sangue di cordone ombelicale, il liquido amniotico ed il tessuto adiposo, e dallo studio delle iPSC, cellule staminali riprogrammate da elementi somatici adulti.

We developed a procedure for growing normal epidermal keratinocyte stem cells isolated from a single punch biopsy of adult human skin in long-term culture. Primary skin epithelial cells were maintained in collagen-coated plates with irradiated human neonatal foreskin fibroblasts (line HPI.1) as a feeder for more than 120 days, approximately 115 population doublings, without signs of replicative senescence. Clonal analysis revealed the presence of holoclones, meroclones, and paraclones. Only emerging colonies with high proliferative potentials and extensive capacities for division (holoclones and meroclones) were subcultured, favoring the expansion of stem cells and progenitors capable of prolonged self-maintenance when subcloned, thus accounting for the prevailing long-term proliferation of the original culture. We found that meroclones included bipotent progenitors capable of generating both keratinocytes and mucin-producing cells. The numbers of these cells were greater after confluence, suggesting that commitment for their differentiation occurred late in the life of a single clone. On a three-dimensional gelatin matrix and on a collagen layer containing the fibroblast feeder, cells isolated from the expansion of holoclones and meroclones formed stratified cohesive layers of keratinocytes that were able to further differentiate, as in normal skin. These results indicate that our procedure will serve as a valuable tool to study expansion of epidermal stem cells as well as the growth mechanisms and cell products associated with their growth and differentiation.

Mesenchymal stem cells (MSC) are currently being investigated in numerous pre-clinical and clinical settings of regenerative medicine. We had previously reported (Revoltella et al., Cell Transplant. 2008; 17, 665-678) that human umbilical cord blood CD133+ stem cells transplanted intravenously (IV) into pre-irradiated nod-scid mice made permanently deaf by ototoxic treatment with kanamycin or intense sound, were able to engraft the cochlea and contribute to inner ear restoration, in vivo. We further investigated here whether human adult MSC derived from either bone marrow or fat (lipid suction), if injected IV to deafened nod-scid mice pre-treated with kanamycin , were able to engraft the damaged cochlea regaining hearing. We tested HLA-DQa1 DNA and three human microsatellites (CODIS) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the Organ of Corti in the cochlea, at 7 and 31 days following MSC transplantation. Histology, immunohistochemistry, and lectin staining confirmed the repair process and stimulation ex novo of morphological recovery in the inner ear, contrasting with the lack of morphological and hearing loss repair in control similarly injured but non-transplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating inner ear with a very limited number of chimaeric cells. Dual color FISH analysis provided evidence of positive engraftment in the inner ear and in other mouse tissues, also revealing small numbers of possible heterokaryons, probably resulting from unstable clones derived from fusion of donor with endogenous cells, up to 2 months following transplantation. Stem cells and differentiation pathways focused PCR arrays favoured to select MSC inducing the best response . These findings support the concept that transplanted MSC migrating to the damaged inner ear area provide conditions for the resumption of a damaged cochlea , emerging as a potential strategy for hearing rehabilitation.

The herbicide dichlobenil selectively causes necrosis of the dorsomedial part of olfactory neuroepithelium (NE) with permanent damage to the underlying mucosa, whereas the lateral part of the olfactory region and the nasal respiratory mucosa remain undamaged. We investigated here whether human umbilical cord blood CD133+ stem cells (HSC) injected intravenously to nod-scid mice pretreated with dichlobenil may engraft the olfactory mucosa and contribute to the regeneration of the damaged NE. We tested HLA-DQα1 DNA and three human microsatellites (Combined DNA Index System) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the olfactory mucosa and bulb, at 7 and 31 days following HSC transplantation. Histology, immunohistochemistry, and lectin staining revealed the morphological recovery of the dorsomedial region of the NE in dichlobenil-treated mice that received HSC, contrasting with the lack of regeneration in similarly injured areas as these remained damaged in control nontransplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating olfactory area with chimeric cells. Electro-olfactograms in response to odorants, to test the functionality of the olfactory NE, confirmed the functional damage of the dorsomedial area in dichlobenil-treated mice and the functional recovery of the same area in transplanted mice. These findings support the concept that transplanted HSC migrating to the damaged olfactory area provide conditions facilitating the recovery from olfactory receptor cell loss. Disclosure of potential conflicts of interest is found at the end of this article.