Rat embryonic stem cells produce fertile offspring through tetraploid complementation.

Pluripotency of embryonic stem cells (ESCs) can be functionally assessed according to the developmental potency. Tetraploid complementation, through which an entire organism is produced from the pluripotent donor cells, is taken as the most stringent test for pluripotency. It remains unclear whether ESCs of other species besides mice can pass this test. Here we show that the rat ESCs derived under 2i (two small molecule inhibitors) conditions at very early passages are able to produce fertile offspring by tetraploid complementation. However, they lose this capacity rapidly during culture due to a nearly complete loss of genomic imprinting. Our findings support that the naïve ground state pluripotency can be captured in rat ESCs but also point to the species-specific differences in its regulation and maintenance, which have implications for the derivation and application of naïve pluripotent stem cells in other species including human.

Human neural stem cells.

'Human neural stem cells' jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human neural stem cells (hNSCs) in China. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for hNSCs, which is applicable to the quality control for hNSCs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that publication of the guideline will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hNSCs for clinical development and therapeutic applications.

Human midbrain dopaminergic progenitors.

Human midbrain dopaminergic progenitors (mDAPs) are one of the most representative cell types in both basic research and clinical applications. However, there are still many challenges for the preparation and quality control of mDAPs, such as the lack of standards. Therefore, the establishment of critical quality attributes and technical specifications for mDAPs is largely needed. "Human midbrain dopaminergic progenitor" jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human mDAPs in China. This standard specifies the technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for human mDAPs, which is applicable to the quality control for human mDAPs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will facilitate the institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human mDAPs for clinical development and therapeutic applications.

Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency.

For the promise of human induced pluripotent stem cells (iPSCs) to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages. Here, we have directly compared the neural-differentiation capacity of human iPSCs and embryonic stem cells (ESCs). We have shown that human iPSCs use the same transcriptional network to generate neuroepithelia and functionally appropriate neuronal types over the same developmental time course as hESCs in response to the same set of morphogens; however, they do it with significantly reduced efficiency and increased variability. These results were consistent across iPSC lines and independent of the set of reprogramming transgenes used to derive iPSCs as well as the presence or absence of reprogramming transgenes in iPSCs. These findings, which show a need for improving differentiation potency of iPSCs, suggest the possibility of employing human iPSCs in pathological studies, therapeutic screening, and autologous cell transplantation.

Cre recombination-mediated cassette exchange for building versatile transgenic human embryonic stem cells lines.

To circumvent the silencing effect of transgene expression in human embryonic stem cells (hESCs), we employed the Cre recombination-mediated cassette exchange strategy to target the silencing-resistant site in the genome. We have identified new loci that sustain transgene expression during stem cell expansion and differentiation to cells representing the three germ layers in vitro and in vivo. The built-in double loxP cassette in the established master hESC lines was specifically replaced by a targeting vector containing the same loxP sites, using the cell-permeable Cre protein transduction method, resulting in successful generation of new hESC lines with constitutive functional gene expression, inducible transgene expression, and lineage-specific reporter gene expression. This strategy and the master cell lines allow for rapid production of transgenic hESC lines in ordinary laboratories.

Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules.

Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these naïve pluripotent cells in regenerative medicine. Determination of the nontarget differentiated populations, which is lacking in the field, is also crucial. Here, we show an efficient differentiation of motor neurons ( approximately 50%) by a simple sequential application of retinoid acid and sonic hedgehog (SHH) in a chemically defined suspension culture. We also discovered that purmorphamine, a small molecule that activates the SHH pathway, could replace SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+, Irx3+, and Pax7-), with the exception of motor neurons (HB9+) and their progenitors (Olig2+). Thus, the directed neural differentiation system with small molecules, even without further purification, will facilitate basic and translational studies using human motoneurons at a minimal cost.

Generation of Bimaternal and Bipaternal Mice from Hypomethylated Haploid ESCs with Imprinting Region Deletions.

Unisexual reproduction is widespread among lower vertebrates, but not in mammals. Deletion of the H19 imprinted region in immature oocytes produced bimaternal mice with defective growth; however, bipaternal reproduction has not been previously achieved in mammals. We found that cultured parthenogenetic and androgenetic haploid embryonic stem cells (haESCs) display DNA hypomethylation resembling that of primordial germ cells. Through MII oocyte injection or sperm coinjection with hypomethylated haploid ESCs carrying specific imprinted region deletions, we obtained live bimaternal and bipaternal mice. Deletion of 3 imprinted regions in parthenogenetic haploid ESCs restored normal growth of fertile bimaternal mice, whereas deletion of 7 imprinted regions in androgenetic haploid ESCs enabled production of live bipaternal mice that died shortly after birth. Phenotypic analyses of organ and body size of these mice support the genetic conflict theory of genomic imprinting. Taken together, our results highlight the factors necessary for crossing same-sex reproduction barriers in mammals.

Human Clinical-Grade Parthenogenetic ESC-Derived Dopaminergic Neurons Recover Locomotive Defects of Nonhuman Primate Models of Parkinson's Disease.

Clinical application of stem cell derivatives requires clinical-grade cells and sufficient preclinical proof of safety and efficacy, preferably in primates. We previously successfully established a clinical-grade human parthenogenetic embryonic stem cell (hPESC) line, but the suitability of its subtype-specific progenies for therapy is not clear. Here, we compared the function of clinical-grade hPESC-derived midbrain dopaminergic (DA) neurons in two canonical protocols in a primate Parkinson's disease (PD) model. We found that the grafts did not form tumors and produced variable but apparent behavioral improvement for at least 24 months in most monkeys in both groups. In addition, a slight DA increase in the striatum correlates with significant functional improvement. These results demonstrated that clinical-grade hPESCs can serve as a reliable source of cells for PD treatment. Our proof-of-concept findings provide preclinical data for China's first ESC-based phase I/IIa clinical study of PD (ClinicalTrials.gov number NCT03119636).

Accreditation of Biosafe Clinical-Grade Human Embryonic Stem Cells According to Chinese Regulations.

Human embryonic stem cells (hESCs) are promising in regenerative medicine. Although several hESC-based clinical trials are under way, a widely accepted standard of clinical-grade cells remains obscure. To attain a completely xeno-free clinical-grade cell line, the system must be free of xenogenic components, the cells must have a comprehensive set of functions, and good manufacturing practice conditions must be used. In this study, following these criteria, we successfully derived two hESC lines, which were thereby considered "clinical-grade embryonic stem cells". In addition to the primary capacity for pluripotency, these two cell lines were efficiently differentiated into various types of clinical-grade progeny. Importantly, the cells were recognized by the National Institutes for Food and Drug Control of China for further eligible accreditation. These data indicate that we have established completely xeno-free clinical-grade hESC lines and their derivatives, which will be valuable for the foundation of an international standard for clinical-grade cells for therapy.

Acute photoreceptor degeneration down-regulates melanopsin expression in adult rat retina.

Melanopsin in retinal ganglion cells plays an important role in mammalian circadian systems. Previous studies indicate melanopsin is responsible for circadian photoentrainment independent of classical rods and cones. However, expression of melanopsin in ganglion cells may be regulated by photoreceptors. In this study, we investigated the effects of N-methyl-N-nitrosourea (MNU)-induced acute photoreceptor degeneration on melanopsin mRNA expression and protein distribution in adult rats. Expression of melanopsin was analyzed 0.5, 1, 5, 7, 13 and 28 days after MNU administration by real-time RT-PCR and immunohistochemistry. MNU-induced gradual degeneration of photoreceptors, and by day 7 most of the photoreceptors were lost. The number of ganglion cells did not change significantly at all time points after MNU injection. In contrast, melanopsin mRNA decreased gradually with the loss of photoreceptors, at the same time pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA levels, which co-express with melanopsin in ganglion cells, were not affected by MNU treatment, indicating decrease of melanopsin mRNA levels is not due to ganglion cell damage. Distribution of melanopsin protein in the dendrites of ganglion cells dramatically decreased with the degeneration of photoreceptors, but its expression in the soma persisted for a long time. Our results suggest that intact photoreceptors maintain the expression of melanopsin and its distribution in ganglion cell dendrites.

Specification of functional neurons and glia from human pluripotent stem cells.

Human pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great promise in regenerative medicine as they are an important source of functional cells for potential cell replacement. These human PSCs, similar to their counterparts of mouse, have the full potential to give rise to any type of cells in the body. However, for the promise to be fulfilled, it is necessary to convert these PSCs into functional specialized cells. Using the developmental principles of neural lineage specification, human ESCs and iPSCs have been effectively differentiated to regional and functional specific neurons and glia, such as striatal gama-aminobutyric acid (GABA)-ergic neurons, spinal motor neurons and myelin sheath forming oligodendrocytes. The human PSCs, in general differentiate after the similar developmental program as that of the mouse: they use the same set of cell signaling to tune the cell fate and they share a conserved transcriptional program that directs the cell fate transition. However, the human PSCs, unlike their counterparts of mouse, tend to respond divergently to the same set of extracellular signals at certain stages of differentiation, which will be a critical consideration to translate the animal model based studies to clinical application.

Directed differentiation of neural-stem cells and subtype-specific neurons from hESCs.

We describe a chemically defined protocol for efficient differentiation of human embryonic stem cells (hESCs) to neural epithelial cells and then to functional spinal motor neurons. This protocol comprises four major steps. Human ESCs are differentiated without morphogens into neuroepithelial cells that form neural tube-like rosettes in the first 2 weeks. The neuroepithelial cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) in the following 2 weeks. These OLIG2 progenitors generate postmitotic, HB9 expressing motoneurons at the fifth week and mature to functional motor neurons thereafter. The protein factor SHH can be replaced by a small molecule purmorphamine in the entire process, which may facilitate potential clinical applications. This protocol has been shown equally effective in generating motor neurons from human induced pluropotent stem (iPS) cells.

Differentiation of spinal motor neurons from pluripotent human stem cells.

We have devised a reproducible protocol by which human embryonic stem cells (hESCs) or inducible pluripotent stem cells (iPSCs) are efficiently differentiated to functional spinal motor neurons. This protocol comprises four major steps. Pluripotent stem cells are induced to form neuroepithelial (NE) cells that form neural tube-like rosettes in the absence of morphogens in the first 2 weeks. The NE cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) or purmorphamine in the next 2 weeks. These progenitor cells further generate post-mitotic, HB9-expressing motoneurons at the 5th week and mature to functional motor neurons thereafter. It typically takes 5 weeks to generate the post-mitotic motoneurons and 8-10 weeks for the production of functional mature motoneurons. In comparison with other methods, our protocol does not use feeder cells, has a minimum dependence on proteins (purmorphamine replacing SHH), has controllable adherent selection and is adaptable for scalable suspension culture.

Differentiation of human oligodendrocytes from pluripotent stem cells.

We have developed a four-part protocol to differentiate human embryonic stem cells (hESCs) to oligodendrocyte progenitor cells (OPCs) according to developmental principles. In the first 2 weeks, hESCs are induced to differentiate into neuroepithelial cells, which form neural tube-like rosettes. In the following 10 d, these neuroepithelial cells are specified to OLIG2-expressing progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH). Upon treatment with fibroblast growth factor 2 (FGF2) for another 10 d, these progenitors convert to OLIG2 and NKX2.2-expressing pre-OPCs. Finally, the pre-OPCs take 8-9 weeks to differentiate into OPCs, which express additional markers of oligodendrocytes, such as SOX10, platelet-derived growth factor receptor alpha (PDGFRalpha) and NG2. The unique aspects of the protocol are the use of FGF2 to promote the differentiation of gliogenic pre-OPCs in the third part and the removal of FGF2 during the transition of pre-OPCs to OPCs. This 3-month differentiation protocol consistently yields OPCs of high purity capable of producing myelin sheaths in vivo.

Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects.

Human embryonic stem cells (hESCs) offer a platform to bridge what we have learned from animal studies to human biology. Using oligodendrocyte differentiation as a model system, we show that sonic hedgehog (SHH)-dependent sequential activation of the transcription factors OLIG2, NKX2.2 and SOX10 is required for sequential specification of ventral spinal OLIG2-expressing progenitors, pre-oligodendrocyte precursor cells (pre-OPCs) and OPCs from hESC-derived neuroepithelia, indicating that a conserved transcriptional network underlies OPC specification in human as in other vertebrates. However, the transition from pre-OPCs to OPCs is protracted. FGF2, which promotes mouse OPC generation, inhibits the transition of pre-OPCs to OPCs by repressing SHH-dependent co-expression of OLIG2 and NKX2.2. Thus, despite the conservation of a similar transcriptional network across vertebrates, human stem/progenitor cells may respond differently to those of other vertebrates to certain extrinsic factors.