Producing Animal Mammary Bioreactor By Cell Transplantation Of Mouse Embryonic Stem-Like Derived Transgenic Precursor Cells

ES cell technology has been the greatest way to product animal mammary gland bioreactor immediately since the first ES cell line was established in 1981. However, ES or ES-like cell line of livestock is difficult to generate and the culture technology which meets the requirement of product animal mammary gland bioreactor could hardly be achieved. In addition, nowadays, the predominant method to generate animal mammary gland bioreactor is to make transgenic animals, which is of great difficulty to implement in practical experiments, and it is definitely a waste of time, money and energy. Therefore, the extension and dissipation of this technique is extremely limited. It is especially important to search a new way to generate animal mammary gland bioreactor quickly.Directed differentiation in vitro serves as one of the most popular research areas in stem cells for decades. Theoretically, more than 200 types of cells could be differentiated from Embryonic Stem (ES) cells in vitro. To date, many types of cells including neural precursor cells, islet cell, keratinocyte, epithelial precursor cell, myocardial cell and germocyte, have been derived from ES cells. It was reported that these ES derived precursor cell or stem cell can further differentiate into receipt related type of cells under microenvironmental induction in vivo after they were transplanted into some special tissue or organ, and survived, integrated in vivo and generated normal physiological function. In addition, some literatures reported that, gene-modified mammary epithelial cells can survive for long time in vivo and expressed foreign gene after they were transplanted into receipt breast tissue. In summary, these results indicated that, it is feasible to produce animal mammary gland bioreactor by cell transplantation of mES-like derived transgenic precursor cells.Consequently, we propose a hypothesis with a model animal of mouse. We suppose that after stably transfected with a mammary-specific expression plasmid containing beta-lactoglobulin (BLG) gene promoter or whey acidic protein (WAP) orβ-casein, the mES derived epithelial precursor cells were transplanted into the mammary gland tissue, if the foreign cells would have survived, integrated in the recipient mammary gland and played normal physiological function there, the interest protein should be secreted into milk by mammary gland, that is, animal mammary gland bioreactor was generated.In this research, mES (at passage three) derived epithelial precursor cells stably transfected with a mammary-specific expression plasmid containing beta-lactoglobulin (BLG) gene promoter and foreign gene PBD-1 (porcineβdefensin 1) were transplanted into the mammary gland tissue during pregnancy. These cells can survive and integrate in the recipient mammary gland under the microenvironmental induction and further differentiate into mammary related type of cells. The main results are as follows:1. 560 collected 3.5- to 4-d old embryos were cultured in DMEM as follows. Method one: MEF (Mouse embryonic fibroblast) cells at different passages (from passage 0 to 5) were used as feeder cells. Method two: the third passage of MEF cells were used as feeder cells and cultured embryos with conditioned medium. On day 5, ICM (Inner cell mass)-outgrowth were isolated from blastocysts and plated on new feeder cells. Three to five days later, these cultures from ICMs have a high ratio of nucleus to cytoplasm, prominent nucleoli, and typical colony with ES cells’ morphological characteristics. They have three dimensional prominent, round or elliptic colonies, with smooth and strongly refractive edges, and have no clear boundaries of cells within a colony. When two methods were compared, Method two is significantly better than Method one on the form ratio of ICM and passage number. These cells were subsequently characterized by RT-PCR and found to be positive for mouse pluripotent embryonic stem cell specific markers Oct-4. In addition, these cells expressed alkaline phosphatase, and staining intensity for SSEA-1 and Oct-4 in brown color while feeder layer failed to stain as control. These cells were diploid chromosome number, and they can differentiate into many types of cells in vitro and in vivo. This validated the pluripotent of mES-like cells obtained in our experiments. Therefore, they can be used as test material later.2. To initiate differentiation, mES-like cell colonies (passage 3) were dispersed to small clumps, containing approximately 50 to 100 cells, thus allowed to grow on mitotically inactivated MEF feeder cells. For the additional 10-13 d of culture, the epithelial-like polygonal cells appeared around the disc-like colonies. They migrated and spreaded until cells were confluent with a typical paving stone-like appearance. These cells were called mES-derived keratinocytes (mES-dK) in this experiment. After the first six passages of co-culture with MEF feeder layers, mES-dK cells were plated onto a new flask without MEF feeder layers. The mES-dK cell line was generated and preservated in CCTCC (China Center for Type Culture Collection). RT-PCR results indicated that, K14, K18, K10, Involucrin and E-cadherin were found to be up regulated in the cells during the differentiation, whereas Oct-4 and K19 were down regulated (compared to pre-differentiation levels). To further assess the expression level during the differentiational process, we also analyzed the expression patterns of K14, K19, Involucrin, and Ecadherin by quantitative real-time PCR during the course of differentiation of mES cells into keratinocyte-like cells. The K14 transcript increased dramatically within 50 d (from mES cells to passage 13) after further differentiation in mES cells committed by removing MEF feeder layers. This level rose to about seven-fold (from passage 13 to 23). On the other hand, expression of K19 RNA rose dramatically during the initial phase (from mES cells to passage 13) and dropped back to a lower level (from passage 13 to 23). It is interesting to note that the expression patterns of Involucrin and E-cadherin transcripts both stayed at the similar level between 13 and 23 passages. Immunocytochemistry showed that almost all mES-dK cells were stained with pCK, K14, and K18, which are epithelial cell surface markers. To check if the keratinocyte-like cells were proliferating, we analyzed the cells by flowing cytometry for BrdU-incorporation and for keratins (K14, K18, and pCK) expression, the majority of cells are positive in both cases (80.5%, 51.3%, 67.4% and 82.7%, respectively). Thus we conclude that the epithelial-like cells are proliferating and presumed it may be an epithelial progenitor lineage.3. The whole cDNA sequence encoding PBD-1 which was amplified by PCR from hepatic cDNA of porcine. BLG gene was amplified from the vector of pcDNA3.1-BLG-HNP-1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP-PBD-1. The recombinant colonies were identified by the methods of restriction enzyme digestion, PCR and sequencing. These results showed that, the mammary gland special expression vector pIRES2-EGFP-BLG-PBD-1 was successfully constructed. The establishment of mammary gland special expression vector pIRES2-EGFP-BLG-PBD-1 serve as a foundation in research on antimicrobial activities and its mechanism of the defensins, and it plays a pivotal role in further research for the establishment of animal mammary bioreactor.4. The pIRES2-EGFP-BLG-PBD-1 plasmid was transfected into mES-dK cells by Lipofectamine 2000. Stably transfected clones were selected using G418 at 2000μg/mL for first 5 weeks and surviving cells were maintained in 1000μg/mL G418. RT-PCR results indicated that, the transgenic mES-dK precursor cells expressed foreign genes PBD-1 and BLG, and the epithelial cell markers, K10, K14, K18 and K19, but did not express Oct-4, Invoclurin or Fibroblast special protein 1(FSP1). Immunocytochemistry showed that majority of mES-dK cells were stained with pCK, K14, and K18, which are used to mark epithelial cell surface.5. Transgenic mES-dK cells were transplanted at different dose into inguinal #4 glands of Kunming white mice. Each animal received a 0.1ml injection of either vehicle (medium) alone or suspension of 5×10~3 or 1×10~7 transgenic mES-dK cells. Survival cells were mainly found in high dose cell graft groups two weeks after transplantation, and five weeks after operation, cells were only found in mid-pregnant group and late-pregnant group with the high dose cell graft. RT-PCR results indicated that, foreign gene PBD-1 was expressed in transplanted mammary gland tissue. Transgenic mES-dK cells further differentiated into EMA~+ or CALLA~+ cells, which were the exclusive surface markers of luminal and myoepithelial cells, respectively, and can be used to separate the cell types to the degree of 98% purity, and the largest percentage of EMA-positive grafted cell was 41.7% and CALLA-positive grafted cell was 28.4% at the 5-week survival period, respectively. In order to determine whether there were some changes on the structure of mammary gland after transplantation with mES-dK cells, HE staining of mammary gland was performed simultaneously. However, there was not significant difference about morphological characteristics of the mammary gland before and after the transplantation.6. We produced a mouse model of animal mammary bioreactor by precursor cell transplantation of transgenic mES-dK cells. Two weeks later, 37.5% (3/8) female transplanted mice had PBD-1 expression in their milk. The pBD-1 concentration was measured with an ELISA kit and the PBD-1 contents in milk reached 514.09±11.47 ng/mL. Western blotting analysis further confirmed that PBD-1 protein (7 ku) was secreted in the milk 2 weeks after transplantation.