技术资料

The shortage of human organs and tissues for transplant has led to significant interest in xenotransplantation of pig tissues for human patients. However,transplantation of pig organs results in an acute immune rejection,leading to death of the organ within minutes. The $\$-1,3-galactosyltransferase (GALT) gene has been knocked out in pigs to reduce rejection,yet additional genes need to be modified to ultimately make pig tissue immunocompatible with humans. The development of pig induced pluripotent stem cells (piPSCs) from GALT knockout (GALT-KO) tissue would provide an excellent cell source for complex genetic manipulations (e.g.,gene targeting) that often require highly robust and proliferative cells. In this report,we generated GALT-KO piPSCs by the overexpression of POU5F1,SOX2,NANOG,LIN28,KLF-4,and C-MYC reprogramming genes. piPSCs showed classical stem cell morphology and characteristics,expressing integrated reprogramming genes in addition to the pluripotent markers AP,SSEA1,and SSEA4. GALT-KO piPSCs were highly proliferative and possessed doubling times and telomerase activity similar to human embryonic stem cells. These results demonstrated successful reprogramming of GALT-KO fibroblasts into GALT-KO piPSCs. GALT-KO piPSCs are potentially an excellent immortal cell source for the generation of pigs with complex genetic modifications for xenotransplantation,somatic cell nuclear transfer,or chimera formation. View Publication

Substrate composition significantly impacts human pluripotent stem cell (hPSC) self-renewal and differentiation,but relatively little is known about the role of endogenously produced extracellular matrix (ECM) components in regulating hPSC fates. Here we identify $\$-5 laminin as a signature ECM component endogenously synthesized by undifferentiated hPSCs cultured on defined substrates. Inducible shRNA knockdown and Cas9-mediated disruption of the LAMA5 gene dramatically reduced hPSC self-renewal and increased apoptosis without affecting the expression of pluripotency markers. Increased self-renewal and survival was restored to wild-type levels by culturing the LAMA5-deficient cells on exogenous laminin-521. Furthermore,treatment of LAMA5-deficient cells with blebbistatin or a ROCK inhibitor partially restored self-renewal and diminished apoptosis. These results demonstrate that endogenous $\$-5 laminin promotes hPSC self-renewal in an autocrine and paracrine manner. This finding has implications for understanding how stem cells dynamically regulate their microenvironment to promote self-renewal and provides guidance for efforts to design substrates for stem cell bioprocessing. View Publication

Thrombocytopenia is a critical problem that occurs in many hematologic diseases,as well as after cancer therapy and radiation exposure. Platelet transfusion is the most commonly used therapy but has limitations of alloimmunization,availability,and expense. Thus,the development of safe,small,molecules to enhance platelet production would be advantageous for the treatment of thrombocytopenia. Herein,we report that an important lipid mediator and a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand called 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)),increases Meg-01 maturation and platelet production. 15d-PGJ(2) also promotes platelet formation from culture-derived mouse and human megakaryocytes and accelerates platelet recovery after in vivo radiation-induced bone marrow injury. Interestingly,the platelet-enhancing effects of 15d-PGJ(2) in Meg-01 cells are independent of PPARgamma,but dependent on reactive oxygen species (ROS) accumulation; treatment with antioxidants such as glutathione ethyl ester (GSH-EE); or N-acetylcysteine (NAC) attenuate 15d-PGJ(2)-induced platelet production. Collectively,these data support the concept that megakaryocyte redox status plays an important role in platelet generation and that small electrophilic molecules may have clinical efficacy for improving platelet numbers in thrombocytopenic patients. View Publication

Embryonic stem cells (ESC) are derived from the inner cell mass (ICM) of preimplantation blastocysts. To date,it has been challenging to establish pluripotent ESC lines for domestic animals,which could be important for biotechnological applications,such as genetic engineering and SCNT,and biomedical research. The aim of this work was to derive and characterise bovine embryonic stem-like cells (bESC) from in vitro-produced bovine blastocysts. Embryos were produced by in vitro fertilization of in vitro-matured oocytes aspirated from abattoir ovaries and cultured in groups of 25 in 50-μL drops of KSOM (Evolve,Zenith Biotech) with 4mgmL(-1) BSA for 7 days until they reached the blastocyst stage (Ross et al.,2009 Reproduction 137,427-437). At that point,the zona pellucida (ZP) was removed using 1mgmL(-1) Pronase (Sigma,St. Louis,MO),and ZP-free blastocysts were washed 6 times in SOF-HEPES. Three derivation approaches were tested: ZP-free whole blastocysts,mechanically isolated ICM,and immunosurgery-derived ICM. In each case,individual blastocysts/ICM were placed in 1 well of a 12-well dish seeded with a monolayer of mouse embryo fibroblasts (MEF) and cultured in mTeSR1 basal medium (without growth factors) supplemented with 20ngmL(-1) FGF2 and 2.5μM IWR1 (CTFR) (Wu et al. 2015 Nature 521,316-321). After 48h,blastocysts/ICM that failed to adhere were physically pressed against the bottom of the culture dish with a 22-gauge needle under a stereoscope to aid attachment. Thereafter,the media was changed daily. Outgrowths (after 6-7 days in culture) were dissociated and passaged using TrypLE and re-seeded in the presence of ROCK inhibitor (Y-27632,10μM) onto newly prepared wells containing MEF. Established bESC lines were cultured on MEF and passaged every 4 to 5 days at a 1:10 split ratio. The bESC lines were characterised by immunofluorescence (IF),RNA-seq,and teratoma formation. The efficiency of cell line derivation (evaluated at passage 3) was similar for the 3 approaches: whole blastocysts (9/16,56.3%),mechanical ICM isolation (7/12,58.3%),and immunosurgical ICM isolation (7/16,43.8%). The bESC were passaged and cultured long-term (more than 15 passages) and were subjected to several rounds of freezing and thawing while retaining their morphology and characteristics. IF analysis showed that long-term cultured bESC expressed the markers SOX2 and OCT4 (pluripotency),but did not express CDX2 (trophectoderm) or GATA6 (primitive endoderm). RNAseq analysis of 2 bESC lines showed that ICM markers (POU5F1,NANOG,SOX2,LIN28B,DNAMT3B,UTF1,SALL4) were expressed (RPKMtextgreater0.4),while trophectoderm markers (CDX2,GATA2,GATA3,FGF4,TFAP2A) and primitive endoderm markers (GATA6,HNF4A) were not expressed (RPKMtextless0.4). Finally,bESC lines (n=2) were able to form teratomas in immunodeficient mice. The teratomas contained tissues representative of the 3 germ lineages and expressed lineage-specific markers (ectoderm: TUJ1,endoderm: FOXA2,and mesoderm: ASM). In conclusion,the culture condition used in this work (CTFR) enables robust derivation and long-term in vitro propagation of pluripotent bESC. View Publication