Marek Maleszewski

This study examined trophectoderm (TE) protrusions covering the inner cell mass (ICM) during blastocyst development. Using fluorescein-labelled latex beads in E3.5 and E4.5 mouse embryos, we assessed the presence of TE protrusions (TP) and their relationship to primitive endoderm (PrE) formation. Beads were endocytosed by outer embryonic cells, enabling visualization of protrusions by confocal microscopy. We observed that TE protrusion coverage of the ICM decreased as blastocyst cell numbers increased, regressing entirely by E4.5. At this stage, no protrusions remained and the ICM exhibited a distinct PrE layer following cell sorting.

Celem niniejszego artykułu jest przegląd danych literaturowych oraz podsumowanie aktualnego stanu wiedzy na temat zwierząt, które mogą stanowić potencjalne źródło narządów do ksentransplantacji. Ksenotransplantacja, rozumiana jako przeszczepianie tkanek lub narządów pomiędzy przedstawicielami różnych gatunków była przedmiotem badań od wielu lat. Pomimo rozważania innych gatunków jako potencjalnych dawców obecnie największe zainteresowanie budzi świnia (Sus domesticus), choć genetycznie różna od człowieka, wykazuje wiele cech sprzyjających temu zastosowaniu. Aby zminimalizować ryzyko odpowiedzi immunologicznej biorcy i odrzutu przeszczepu stosuje się modyfikacje genetyczne zwierząt. Rozwój technik genetyki molekularnej umożliwił szybkie i precyzyjne uzyskiwanie zmodyfikowanych zwierząt, których narządy są bardziej kompatybilne z ludzkim układem immunologicznym. Ksenotransplantacja postrzegana jest jako jedno z potencjalnych rozwiązań narastającego problemu niedoboru narządów do przeszczepów, związanego ze starzeniem się społeczeństw oraz wzrostem populacji. Praca ta analizuje zalety i ograniczenia ksenotransplantacji uwzględniając zarówno aspekty techniczne jaki i etyczne. Przedstawiono dotychczasowy rozwój tej dziedziny, w tym historie badań, potencjalne komplikacje związane z ksenoprzeszczepami, szczegóły dotyczące modyfikacji zwierząt oraz omówiono najnowsze osiągnięcia, w tym przeprowadzone już eksperymentalne przeszczepy u ludzi.

Liver transplantation is the only curative option for end-stage liver disease and is necessary for an increasing number of patients with advanced primary or secondary liver cancer. Many patient groups can benefit from this treatment, however the shortage of liver grafts remains an unsolved problem. Liver bioengineering offers a promising method for expanding the donor pool through the production of acellular scaffolds that can be seeded with recipient cells. Decellularization protocols involve the removal of cells using various chemical, physical, and enzymatic steps to create a collagenous network that provides support for introduced cells and future vascular and biliary beds. However, the removal of the cells causes varying degrees of matrix damage, that can affect cell seeding and future organ performance. The main objective of this review is to present the existing techniques of producing decellularized livers, with an emphasis on the assessment and definition of acellularity. Decellularization agents are discussed, and the standard process of acellular matrix production is evaluated. We also introduce the concept of the stepwise assessment of the matrix during decellularization through decellularization cycles. This method may lead to shorter detergent exposure times and less scaffold damage. The introduction of apoptosis induction in the field of organ engineering may provide a valuable alternative to existing long perfusion protocols, which lead to significant matrix damage. A thorough understanding of the decellularization process and the action of the various factors influencing the final composition of the scaffold is essential to produce a biocompatible matrix, which can be the basis for further studies regarding recellularization and retransplantation.

Aggregates of two mouse embryos produce viable offspring of normal size, indicating that there are mechanisms in the embryo that can downregulate their size to the size of the corresponding normal (single) embryos. Very little is known about the mechanisms controlling compensation for increased preimplantation size. Also, it is still elusive when exactly during development chimeric embryos regulate their size. Here, we determined the exact period of size regulation in chimeras. Using a chimeric embryo produced by aggregating two 8-cell stage embryos, we revealed that size regulation initiates shortly after implantation (E5.5) and ends with the start of gastrulation (E7.5). Importantly, processes that regulate cell number in chimeric embryos do not disturb morphogenesis, so that the formation of the proamniotic cavity occurs in parallel with size regulation.

Here we present our results regarding the role of E-cadherin (CDH1, cadherin 1) in the specification of embryonic stem cells (ESCs) in the embryo environment. It has been previously shown that, when forming embryoid bodies with wild-type ESCs, ESCs lacking a functional copy of the gene encoding the adhesive protein E-cadherin (Ecad ^−/− ) preferably sort out to the outside compartment, thus forming the primitive endoderm (PrE) lineage. However, little or no information is available regarding the dynamics of Ecad ^−/− cells in the actual blastocyst, and so the aim of this work was to determine in which of the three blastocyst cell lines - trophoblast (TE), epiblast (Epi), or PrE - Ecad ^−/− cells would be located in the embryo environment. For this purpose we injected ESCs into embryos at various stages of pre-implantation development. We used a H2B-GFP cell line expressing histone H2B conjugated with green fluorescent protein (H2BEGFP), as well as a Ecad ^−/− cell line, in which cells exhibit weaker adhesive properties than wild-type Esc because of their allelic deficiency in the locus encoding cadherin CDH1. We have demonstrated that these cells exhibit a trend to locate in the TE and much less frequently in the Epi, but never in the PrE. We propose that this may be due to differences in the expression of genes characteristic of these cell lines within the Ecad ^−/− cell colony.

During preimplantation development, mammalian embryo cells (blastomeres) cleave, gradually losing their potencies and differentiating into three primary cell lineages: epiblast (EPI), trophectoderm (TE), and primitive endoderm (PE). The exact moment at which cells begin to vary in their potency for multilineage differentiation still remains unknown. We sought to answer the question of whether single cells isolated from 2- and 4-cell embryos differ in their ability to generate the progenitors and cells of blastocyst lineages. We revealed that twins were often able to develop into blastocysts containing inner cell masses (ICMs) with PE and EPI cells. Despite their capacity to create a blastocyst, the twins differed in their ability to produce EPI, PE, and TE cell lineages. In contrast, quadruplets rarely formed normal blastocysts, but instead developed into blastocysts with ICMs composed of only one cell lineage or completely devoid of an ICM altogether. We also showed that quadruplets have unequal capacities to differentiate into TE, PE, and EPI lineages. These findings could explain the difficulty of creating monozygotic twins and quadruplets from 2- and 4-cell stage mouse embryos.

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.

During preimplantation mouse development stages, emerging pluripotent epiblast (Epi) and extraembryonic primitive endoderm (PrE) cells are first distributed in the blastocyst in a “salt-and-pepper” manner before they segregate into separate layers. As a result of segregation, PrE cells become localised on the surface of the inner cell mass (ICM), and the Epi is enclosed by the PrE on one side and by the trophectoderm on the other. During later development, a subpopulation of PrE cells migrates away from the ICM and forms the parietal endoderm (PE), while cells remaining in contact with the Epi form the visceral endoderm (VE). Here, we asked: what are the mechanisms mediating Epi and PrE cell segregation and the subsequent VE vs PE specification? Differences in cell adhesion have been proposed; however, we demonstrate that the levels of plasma membrane-bound E-cadherin (CDH1, cadherin 1) in Epi and PrE cells only differ after the segregation of these lineages within the ICM. Moreover, manipulating E-cadherin levels did not affect lineage specification or segregation, thus failing to confirm its role during these processes. Rather, we report changes in E-cadherin localisation during later PrE-to-PE transition which are accompanied by the presence of Vimentin and Twist, supporting the hypothesis that an epithelial-to-mesenchymal transition process occurs in the mouse peri-implantation blastocyst.

In order to ensure successful development, cells of the early mammalian embryo must differentiate to either trophectoderm (TE) or inner cell mass (ICM), followed by epiblast (EPI) or primitive endoderm (PE) specification within the ICM. Here, we deciphered the mechanism that assures the correct order of these sequential cell fate decisions. We revealed that TE-deprived ICMs derived from 32-cell blastocysts are still able to reconstruct TE during in vitro culture, confirming totipotency of ICM cells at this stage. ICMs isolated from more advanced blastocysts no longer retain totipotency, failing to form TE and generating PE on their surface. We demonstrated that the transition from full potency to lineage priming is prevented by inhibition of the FGF/MAPK signalling pathway. Moreover, we found that after this first restriction step, ICM cells still retain fate flexibility, manifested by ability to convert their fate into an alternative lineage (PE towards EPI and vice versa), until peri-implantation stage.