Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy / 生物工程学报

Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.

Modeling human pregastrulation development by 3D culture of blastoids generated from primed-to-naïve transitioning intermediates

Human pluripotent stem cells provide an inexhaustible model to study human embryogenesis in vitro. Recent studies have provided diverse models to generate human blastoids by self-organization of different pluripotent stem cells or somatic reprogramming intermediates. However, whether blastoids can be generated from other cell types or whether they can recapitulate postimplantation development in vitro is unknown. Here, we develop a strategy to generate human blastoids from heterogeneous intermediates with epiblast, trophectoderm, and primitive endoderm signatures of the primed-to-naïve conversion process, which resemble natural blastocysts in morphological architecture, composition of cell lineages, transcriptome, and lineage differentiation potential. In addition, these blastoids reflect many features of human peri-implantation and pregastrulation development when further cultured in an in vitro 3D culture system. In summary, our study provides an alternative strategy to generate human blastoids and offers insights into human early embryogenesis by modeling peri- and postimplantation development in vitro.

The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.

Multiplexed single-cell transcriptome analysis reveals molecular characteristics of monkey pluripotent stem cell lines / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Efforts have been made to establish various human pluripotent stem cell lines. However, such methods have not yet been duplicated in non-human primate cells. Here, we introduce a multiplexed single-cell sequencing technique to profile the molecular features of monkey pluripotent stem cells in published culture conditions. The results demonstrate suboptimized maintenance of pluripotency and show that the selected signaling pathways for resetting human stem cells can also be interpreted for establishing monkey cell lines. Overall, this work legitimates the translation of novel human cell line culture conditions to monkey cells and provides guidance for exploring chemical cocktails for monkey stem cell line derivation.

LIN28 coordinately promotes nucleolar/ribosomal functions and represses the 2C-like transcriptional program in pluripotent stem cells

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

Caracterización y marcación de células germinales primordiales / Characterization and marking of primordial germ cells

Las células germinales primordiales (CGP) son los precursores de los ovocitos y espermatocitos. Se caracterizan por ser las únicas capaces de retener verdaderamente la capacidad de desarrollo pluripotencial luego de la gastrulación. Estas células provienen del epiblasto, diferenciándose del resto de las células somáticas gracias a señales emitidas por el ectodermo extraembrionario y endodermo visceral tempranamente a partir de los días 6,0 a 7,5 dpc. en la especie murina. Luego migran a través de intestino primitivo y mesenterio dorsal hasta llegar a las crestas genitales alrededor de los 10,5 dpc. Las células germinales primordiales tienen una variada morfología y expresión génica, por lo tanto presentan distintos marcadores según el estadío en el que se encuentren. Dentro de los marcadores más estudiados y caracterizados se encuentran fragillis, c-Kit, Stella, DAZ, Vasa, GCNA1, Blimp1 y SSEA-1.

Primordial germ cells (PGC) are the precursors of oocytes and spermatocytes. They are characterized by being the only ones capable of accurately retaining pluripotent developmental ability after gastrulation. These cells come from the epiblast, they differ from somatic cells by signals from the extra-embryonic ectoderm and visceral endoderm, starting from day 6.0 to 7.5 dpc in murine species. Then migrate through the primitive gut and dorsal mesentery to reach genital ridges around 10.5 dpc. The primordial germ cells have a varied morphology and gene expression therefore have different markers according to the stage where they are. Among the most studied and characterized markers are Fragilis, c-Kit, Stella, DAZ, Vasa, GCNA1, Blimp1 and SSEA-1.

Pluripotent lineage of CD133 stem cells isolated from human skin samples.

Skin stem cells are very important in cosmetics, pharmacological and regenerative medicine and burn cases. Foreskin samples surgically removed after circumcision from boys below 7 years were collected and primary epidermal cells were prepared by enzymatic and mechanical tituration method. Selecting CD133 (prominin-1) multipotent stem cell marker, enriched stem cells were analyzed by MACS using CD133 antibodies conjugated with magnetic beads. CD133 positive and negative cells with specific skin stem cells markers like - CD34 (Universal stem cells marker), CD29 (integrin beta-1) and CD49f (integrin alpha-6) immunophenotypes were screened and sorted in flowcytometer. Further the expression of four embryonic genes or transcription factors of pluripotent stem cells were analyzed for pluripotent character of sorted cells. It was found that skin stem cell markers associated with CD133 cells, differentially expressed CD34, CD29 and CD49f immunophenotyes on both positive and negative CD133 cells in FACS analysis. The embryonic stem cell markers (induced pluripotent stem cell markers) like Oct4, SOX2, Notch-2 and K19 genes were expressed in CD133 positive epidermal cells. It is therefore evident that foreskin derived epidermal stem cells showed pluripotent or multipotent nature. This finding opens up avenues for new uses of these stem cells for direct cell seeding in wound healing, surgical suturing and drug screening.