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1.
Cells ; 12(5)2023 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-36899816

RESUMO

Subcutaneous adipose tissue is an excellent source of mesenchymal stem cells (ADSCs), which can be used in cell therapies as an active substance in advanced therapy medicinal products (ATMPs). Because of the short shelf-life of ATMPs and the time needed to obtain the results of microbiological analysis, the final product is often administered to the patient before sterility is confirmed. Because the tissue used for cell isolation is not sterilized to maintain cell viability, controlling and ensuring microbiological purity at all stages of production is crucial. This study presents the results of monitoring the contamination incidence during ADSC-based ATMP manufacturing over two years. It was found that more than 40% of lipoaspirates were contaminated with thirteen different microorganisms, which were identified as being physiological flora from human skin. Such contamination was successfully eliminated from the final ATMPs through the implementation of additional microbiological monitoring and decontamination steps at various stages of production. Environmental monitoring revealed incidental bacterial or fungal growth, which did not result in any product contamination and was reduced thanks to an effective quality assurance system. To conclude, the tissue used for ADSC-based ATMP manufacturing should be considered contaminated; therefore, good manufacturing practices specific to this type of product must be elaborated and implemented by the manufacturer and the clinic in order to obtain a sterile product.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco , Humanos , Preparações Farmacêuticas
2.
Biol Reprod ; 102(2): 499-510, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31511860

RESUMO

The full-term development of the xenogeneic embryo in the uterus of the mother of different species is very restricted and can occur only in certain groups of closely related mammals. In the case of mouse ↔ rat chimeras, the interspecific uterine barrier is less hostile to interspecific chimeric fetuses. In current work, we tested the development of mouse and rat fetuses in uteri of females of the opposite species. We created chimeric mouse ↔ rat blastocysts by injection of mouse embryonic stem cells (ESCs) into eight-cell rat embryos and rat ESCs into eight-cell mouse embryos. Chimeras were transferred to the foster mothers of the opposite species. Despite a huge number of transferred embryos (>1000 in total for both variants), only one live fetus derived solely from the mouse ESCs was isolated at E13.5 from the rat uterus. All other fetuses and newborns were chimeric or were built only from the cells of the recipient embryo. We examined the possible reason for such an outcome and found that the xenogeneic fetuses are eliminated at the perigastrulation stage of development. Thus, we conclude that in the rat ↔ mouse combination even when extraembryonic tissues of the chimeric embryo are composed solely of the cells of the same species as the female to which embryos are transferred, the full-term development of the pure xenogeneic fetus is very unlikely.


Assuntos
Implantação do Embrião/fisiologia , Transferência Embrionária/veterinária , Desenvolvimento Embrionário/fisiologia , Útero/fisiologia , Animais , Quimera , Feminino , Camundongos , Ratos
3.
Dev Biol ; 427(1): 106-120, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28499799

RESUMO

In order to examine interactions between cells originating from different species during embryonic development we constructed interspecific mouse↔rat chimaeras by aggregation of 8-cell embryos. Embryos of both species expressed different fluorescent markers (eGFP and DsRed), which enabled us to follow the fate of both components from the moment of aggregation until adulthood. We revealed that in majority of embryos the blastocyst cavity appeared inside the group of rat cells, while the mouse component was allocated to the deeper layer of the inner cell mass and to the polar trophectoderm. However, due to rearrangement of all cells and selective elimination of rat cells, shortly before implantation all primary lineages became chimaeric. Moreover, despite the fact that rat cells were always present in the mural trophectoderm, majority of mouse↔rat chimaeric blastocysts implanted in mouse uterus, and out of those 46% developed into foetuses and pups, half of which were chimaeric. In contrast to mural trophectoderm, polar trophectoderm derivatives, i.e. the placentae of all chimaeras were exclusively of mouse origin. This strongly suggests that the successful postimplantation development of chimaeras is enabled by gradual elimination of xenogeneic cells from the nascent placenta. The size of chimaeric newborns was within the limits of control mouse neonates. The rat component located preferentially in the anterior part of the body, where it contributed mainly to the neural tube. Our observations indicate that although chimaeric animals were able to reach adulthood, high contribution of rat cells tended to diminish their viability.


Assuntos
Quimera/embriologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Animais , Animais Recém-Nascidos , Blastocisto/citologia , Blastocisto/metabolismo , Agregação Celular/genética , Linhagem da Célula/genética , Quimera/genética , Quimera/crescimento & desenvolvimento , Implantação do Embrião , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Microscopia de Fluorescência , Gravidez , Ratos Transgênicos , Ratos Wistar , Especificidade da Espécie , Imagem com Lapso de Tempo/métodos
4.
PLoS One ; 12(3): e0175032, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28362853

RESUMO

The mouse preimplantation embryo generates the precursors of trophectoderm (TE) and inner cell mass (ICM) during the 8- to 16-cell stage transition, when the apico-basal polarized blastomeres undergo divisions that give rise to cells with different fate. Asymmetric segregation of polar domain at 8-16 cell division generate two cell types, polar cells which adopt an outer position and develop in TE and apolar cells which are allocated to inner position as the precursors of ICM. It is still not know when the blastomeres of 8-cell stage start to be determined to undergo asymmetric division. Here, we analyze the frequency of symmetric and asymmetric divisions of blastomeres isolated from 8-cell stage embryo before and after compaction. Using p-Ezrin as the polarity marker we found that size of blastomeres in 2/16 pairs cannot be used as a criterion for distinguishing symmetric and asymmetric divisions. Our results showed that at early 8-cell stage, before any visible signs of cortical polarity, a subset of blastomeres had been already predestined to divide asymmetrically. We also showed that almost all of 8-cell stage blastomeres isolated from compacted embryo divide asymmetrically, whereas in intact embryos, the frequency of asymmetric divisions is significantly lower. Therefore we conclude that in intact embryo the frequency of symmetric and asymmetric division is regulated by cell-cell interactions.


Assuntos
Blastômeros/citologia , Embrião de Mamíferos/citologia , Animais , Blastocisto , Blastômeros/metabolismo , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Comunicação Celular/genética , Comunicação Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Camundongos
5.
Int J Dev Biol ; 61(1-2): 1-3, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28287240

RESUMO

Professor Andrzej Krzysztof Tarkowski passed away last September (2016) at the age of 83. His findings, have become indispensable tools for immunological, genetic, and oncological studies, as well as for generating transgenic animals which are instrumental for studying gene function in living animals. His work and discoveries provided a tremendous input to the contemporary developmental biology of mammals.


Assuntos
Biologia do Desenvolvimento/história , História do Século XX , História do Século XXI , Polônia
6.
Mech Dev ; 141: 40-50, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27345419

RESUMO

During mouse embryogenesis initial specification of the cell fates depends on the type of division during 8- to 16- and 16- to 32-cell stage transition. A conservative division of a blastomere creates two polar outer daughter cells, which are precursors of the trophectoderm (TE), whereas a differentiative division gives rise to a polar outer cell and an apolar inner (the presumptive inner cell mass - ICM) cell. We hypothesize that the type of division may depend on the interactions between blastomeres of the embryo. To investigate whether modification of these interactions influences divisions, we analyzed the pattern of blastomere division and cell lineage specification in chimeric embryos obtained by injection of a different number of mouse embryonic stem cells (ESCs) into 8-cell embryos. As the ESCs populate only the ICM of the resulting chimeric blastocysts, they emulated in our model additional inner cells. We found that introduction of ESCs decreased the number of inner, apolar blastomeres at the 8- to 16-cell stage transition and reduced the number of ICM cells of host embryo-origin during formation of the blastocyst. Moreover, we showed that the proportion of inner blastomeres and their fate (EPI or PE) in chimeric blastocysts was dependent on the number of ESCs injected. Our results suggest the existence of a regulative mechanism, which links number of inner cells with a proportion of conservative vs. differentiative blastomere divisions during the cleavage and thus dictates their developmental fate.


Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/transplante , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Embrião de Mamíferos , Camundongos
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