Polpa dentária vira espermatozóide humano em testículo de camundongo

Dois cientistas russos - Irina e Alexandre Kerkis - "importados" por Darcy Ribeiro para Campos de Goytacazes, que depois foram para a USp e terminaram na Clínica Abdelmassih em São Paulo (Irina também é do Instituto Butantan de SP), conseguiram uma proeza: identificar células-tronco na polpa de dente de leite de um doador macho humano, isolá-las, injetá-las no testículo de um camundongo e depois extrair dele espermatozóides humanos produzidos pelo animal.

Esquisito, porém esperto. Dois anos atrás a dupla já havia causado espanto ao conseguir produzir tanto óvulos quanto espermatozóides maduros in vitro (fora do corpo) a partir de células-tronco embrionárias (CTEs) de camundongo. O estudo abria a possibilidade inquietante de produzir óvulos (gametas por definição femininos) a partir de células masculinas. Comentei o assunto na época em uma coluna (que você pode ler aqui).

Soube da nova proeza com os dentes de leite e os roedores por meio de um press release (comunicado para jornalistas), que alerta tratar-se de pesquisa básica, ainda distante de aplicação clínica, mas que não deixa de falar em "esperança para homens inférteis". É o tipo da mensagem dupla que costuma acompanhar a promoção de certos estudos biotecnológicos.

Ora, em primeiro lugar dentes de leite só existem em meninos, não em homens adultos. Talvez se encontrem células-tronco com esse potencial nos dentes ou noutro tecido de um adulto infértil, mas não há garantia disso. A esperança é para lá de longínqua.

Depois, quem gostaria de engravidar com espermatozóides produzidos num roedor? Além da aversão simbólica, há questões de biossegurança, como a possibilidade de esse material ser infectado por vírus endógenos do animal.

O comunicado tem outros problemas, como chamar o Sunday Times de revista (é um jornal, que publicou reportagem sobre a pesquisa) e camundongo de rato (são espécies e gêneros diferentes, Mus musculus e Rattus norvegicus). Mas tem o mérito de reproduzir o abstract (resumo em inglês) do trabalho apresentado pelos Kerkis no Congresso Europeu de Reprodução Humana em Barcelona:


Human sperm cells yielded by adult stem cells transplantation into mouse testis

Kerkis, A.; Fonseca, SAS.; Lavagnolli, TMC.; Serafim, RC.; Abdelmassih, S.; Santos, EJC.; Pereira, VS.;  Abdelmassih, R.; Irina Kerkis.

Stem Cell Laboratory, Roger Abdelmassih Human Reproduction Clinic and Research Center; Laboratory of Genetics, Butantan Institute, Sao Paulo, SP, Brazil.

Introduction: Two cellular lineages, the germline and the soma, are involved in the process of spermatogenesis. Diploid cells, which are committed to originating germ cells (GC), proliferate and differentiate, undergo meiosis and further maturate as sperm. This multi-step process is frequently prone to errors, which leads to defective sperm formation and as a result to male infertility. Different therapeutic approaches are being developed using animal models for male infertility treatment, such as the transplantation of spermatogonial stem cells (SSC) from fertile donors to the testis of infertile ones to obtain donor-derived spermatogenesis. Another approach involves the in vitro derivation of GC from adult stem cells  (ASC). Here we extend both of these techniques aiming at the obtainment of patient-matched sperm cells from human adult stem cells using mouse testis as a conditioning environment. Material and Methods: Multipotent human immature dental pulp stem cells (hIDPSC) were isolated and reviously characterized by our group. At the present study female and male hIDPSC were used. RT-PCR analysis was performed in undifferentiated hIDPSC for Oct4, Nanog, keratin (CK18), vimentin, integrin subunit b1 and deleted in azoospermia (Daz cluster gene). The cell suspension (105 cells) was stained with Vybrant CM-DiI and injected into the testis of fertile mice. Control mice were injected with physiologic solution. The mice were killed 3, 5 and 9 days after hIDPSC injection. Thin whole-testis frozen sections (5µm) were prepared. Presence of hIDPSC in mouse testis as detected by Vybrant CM-DiI and by Fluorescent in situ hybridization (FISH) for human sex chromosomes. The homing of hIDPSC, within mouse testis, was analyzed by confocal microscopy.

Results: Our data demonstrated that undifferentiated hIDPSC express Oct-4 and Nanog (markers of primordial GC), as well as markers detected in germinal epithelium (Vimentin, CK18 and integrin subunit b1) and DAZ, gene expressed in GC. After hIDPSC transplantation into mouse testis fluorescent signals were observed within transversal sections of seminiferous tubules (ST). At day 3 after transplantation, hIDPSC were detected mainly in cell compartment, where Leyding and Sertoli cells were located. At days 5 and 9, these cells formed fluorescently labelled clusters similar to defined GC associations within mouse ST, which contain the cells morphologically similar to spermatogonia and to human sperm, observed in central part of the lumen. In all experiments these fluorescent cells have been seen only in a few ST. Control mouse ST (without hIDPSC) did not show any fluorescence. FISH analysis for human sex chromosomes confirmed our findings of hIDPSC haploidization, during the process of GC differentiation.

Conclusions: We showed that undifferentiated hIDPSC already express several genes involved in GC development and differentiation, which suggest their GC commitment. When transplanted in mouse testis, hIDPSC underwent differentiation following sequential order of mouse seminiferous epithelium. Our data indicate that the mouse ST environment influences the process of hIDPSC differentiation, yielding human sperm cells, although at a low efficiency. In this manner, the use of an animal ST environment would be a tool for human ASC differentiation in order to achieve human donor-derived spermatogenesis.

The present work was supported by the Roger Abdelmassih Human Reproduction Clinic and Research Center