GMP-compliant iPS cell lines show widespread plasticity in a new set of differentiation workflows for cell replacement and cancer immunotherapy.

Cell therapeutic applications based on induced pluripotent stem cells (iPSCs) appear highly promising and challenging at the same time. Good manufacturing practice (GMP) regulations impose necessary yet demanding requirements for quality and consistency when manufacturing iPSCs and their differentiated progeny. Given the scarcity of accessible GMP iPSC lines, we have established a corresponding production workflow to generate the first set of compliant cell banks. Hence, these lines met a comprehensive set of release specifications and, for instance, displayed a low overall mutation load reflecting their neonatal origin, cord blood. Based on these iPSC lines, we have furthermore developed a set of GMP-compatible workflows enabling improved gene targeting at strongly enhanced efficiencies and directed differentiation into critical cell types: A new protocol for the generation of retinal pigment epithelium (RPE) features a high degree of simplicity and efficiency. Mesenchymal stromal cells (MSCs) derived from iPSCs displayed outstanding expansion capacity. A fully optimized cardiomyocyte differentiation protocol was characterized by a particularly high batch-to-batch consistency at purities above 95%. Finally, we introduce a universal immune cell induction platform that converts iPSCs into multipotent precursor cells. These hematopoietic precursors could selectively be stimulated to become macrophages, T cells, or natural killer (NK) cells. A switch in culture conditions upon NK-cell differentiation induced a several thousand-fold expansion, which opens up perspectives for upscaling this key cell type in a feeder cell-independent approach. Taken together, these cell lines and improved manipulation platforms will have broad utility in cell therapy as well as in basic research.

Directed Differentiation of Human Pluripotent Stem Cells to Cytotrophoblast and Syncytiotrophoblast.

Human pluripotent stem cells (hPSCs) form an ideal system to study the formation of placental cells, from an undifferentiated human embryonic stem cell state. The conventional human in vitro model systems to study the human placenta cannot be employed for understanding placental dysfunctions or the development of specialized placental cell types. Hence, human PSCs make an ideal model system to study human placental development and disorders. Here, we describe an efficient and validated protocol to reproducibly study the formation of human cytotrophoblasts (CTBs) and syncytiotrophoblast (STBs) from undifferentiated hPSCs. CTBs are the trophoblast stem cells that can differentiate into specialized placental cell types such as STBs. The multinucleated STB plays vital role in the exchange of nutrients and gases across the placenta and secretes several hormones during pregnancy, such as human chorionic gonadotropin ß (hCGß). Here we describe two methods of seeding the hPSCs: chemical (clumps method) and enzymatic methods (single cells) to differentiate them to CTB and STB, activating BMP (B) signaling and inhibiting ACTIVIN/NODAL and FGF signaling pathways (2i), thus naming our protocol as "B2i" (Sudheer et al., Stem Cells Dev 21:2987-3000, 2012). This protocol forms the perfect model system for understanding in vitro placentation, modeling diseases arising from abnormal placentation that cause complications such as miscarriage, preeclampsia or intrauterine growth restriction (IUGR), and drug discovery for placental disorders.

Human ES Cell Culture Conditions Fail to Preserve the Mouse Epiblast State.

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) are the pluripotent stem cells (PSCs), derived from the inner cell mass (ICM) of preimplantation embryos at embryonic day 3.5 (E3.5) and postimplantation embryos at E5.5-E7.5, respectively. Depending on their environment, PSCs can exist in the so-called naïve (ESCs) or primed (EpiSCs) states. Exposure to EpiSC or human ESC (hESC) culture condition can convert mESCs towards an EpiSC-like state. Here, we show that the undifferentiated epiblast state is however not stabilized in a sustained manner when exposing mESCs to hESC or EpiSC culture condition. Rather, prolonged exposure to EpiSC condition promotes a transition to a primitive streak- (PS-) like state via an unbiased epiblast-like intermediate. We show that the Brachyury-positive PS-like state is likely promoted by endogenous WNT signaling, highlighting a possible species difference between mouse epiblast-like stem cells and human Embryonic Stem Cells.

Importance of Long Non-coding RNAs in the Development and Disease of Skeletal Muscle and Cardiovascular Lineages.

The early mammalian embryo is characterized by the presence of three germ layers-the outer ectoderm, middle mesoderm and inner endoderm. The mesoderm is organized into paraxial, intermediate and lateral plate mesoderm. The musculature, vasculature and heart of the adult body are the major derivatives of mesoderm. Tracing back the developmental process to generate these specialized tissues has sparked much interest in the field of regenerative medicine focusing on generating specialized tissues to treat patients with degenerative diseases. Several Long Non-Coding RNAs (lncRNAs) have been identified as regulators of development, proliferation and differentiation of various tissues of mesodermal origin. A better understanding of lncRNAs that can regulate the development of these tissues will open potential avenues for their therapeutic utility and enhance our knowledge about disease progression and development. In this review, we aim to summarize the functions and mechanisms of lncRNAs regulating the early mesoderm differentiation, development and homeostasis of skeletal muscle and cardiovascular system with an emphasis on their therapeutic potential.

The quest for pluripotency: a comparative analysis across mammalian species.

Pluripotency is the developmental potential of a cell to give rise to all the cells in the three embryonic germ layers, including germline cells. Pluripotent stem cells (PSCs) can be embryonic, germ cell or somatic cell in origin and can adopt alternative states of pluripotency: naïve or primed. Although several reports have described the differentiation of PSCs to extra-embryonic lineages, such as primitive endoderm and trophectoderm, this is still debated among scientists in the field. In this review, we integrate the recent findings on pluripotency among mammals, alternative states of pluripotency, signalling pathways associated with maintaining pluripotency and the nature of PSCs derived from various mammals. PSCs from humans and mouse have been the most extensively studied. In other mammalian species, more research is required for understanding the optimum in vitro conditions required for either achieving pluripotency or preservation of distinct pluripotent states. A comparative high-throughput analysis of PSCs of genes expressed in naïve or primed states of humans, nonhuman primates (NHP) and rodents, based on publicly available datasets revealed the probable prominence of seven signalling pathways common among these species, irrespective of the states of pluripotency. We conclude by highlighting some of the unresolved questions and future directions of research on pluripotency in mammals.

Antagonistic Activities of Sox2 and Brachyury Control the Fate Choice of Neuro-Mesodermal Progenitors.

The spinal cord and mesodermal tissues of the trunk such as the vertebral column and skeletal musculature derive from neuro-mesodermal progenitors (NMPs). Sox2, Brachyury (T), and Tbx6 have been correlated with NMP potency and lineage choice; however, their exact role and interaction in these processes have not yet been revealed. Here we present a global analysis of NMPs and their descending lineages performed on purified cells from embryonic day 8.5 wild-type and mutant embryos. We show that T, cooperatively with WNT signaling, controls the progenitor state and the switch toward the mesodermal fate. Sox2 acts antagonistically and promotes neural development. T is also involved in remodeling the chromatin for mesodermal development. Tbx6 reinforces the mesodermal fate choice, represses the progenitor state, and confers paraxial fate commitment. Our findings refine previous models and establish molecular principles underlying mammalian trunk development, comprising NMP maintenance, lineage choice, and mesoderm formation.

Different Concentrations of FGF Ligands, FGF2 or FGF8 Determine Distinct States of WNT-Induced Presomitic Mesoderm.

Presomitic mesoderm (PSM) cells are the precursors of the somites, which flank both sides of the neural tube and give rise to the musculo-skeletal system shaping the vertebrate body. WNT and FGF signaling control the formation of both the PSM and the somites and show a graded distribution with highest levels in the posterior PSM. We have used reporters for the mesoderm/PSM control genes T, Tbx6, and Msgn1 to investigate the differentiation of mouse ESCs from the naïve state via EpiSCs to PSM cells. Here we show that the activation of WNT signaling by CHIR99021 (CH) in combination with FGF ligand induces embryo-like PSM at high efficiency. By varying the FGF ligand concentration, the state of PSM cells formed can be altered. High FGF concentration supports posterior PSM formation, whereas low FGF generates anterior/differentiating PSM, in line with in vivo data. Furthermore, the level of Msgn1 expression depends on the FGF ligand concentration. We also show that Activin/Nodal signaling inhibits CH-mediated PSM induction in EpiSCs, without affecting T-expression. Inversely, Activin/Nodal inhibition enhances PSM induction by WNT/high FGF signaling. The ability to generate PSM cells of either posterior or anterior PSM identity with high efficiency in vitro will promote the investigation of the gene regulatory networks controlling the formation of nascent PSM cells and their switch to differentiating/somitic paraxial mesoderm. Stem Cells 2016;34:1790-1800.

FGF inhibition directs BMP4-mediated differentiation of human embryonic stem cells to syncytiotrophoblast.

Bone morphogenetic protein (BMP) signaling is known to support differentiation of human embryonic stem cells (hESCs) into mesoderm and extraembryonic lineages, whereas other signaling pathways can largely influence this lineage specification. Here, we set out to reinvestigate the influence of ACTIVIN/NODAL and fibroblast growth factor (FGF) pathways on the lineage choices made by hESCs during BMP4-driven differentiation. We show that BMP activation, coupled with inhibition of both ACTIVIN/NODAL and FGF signaling, induces differentiation of hESCs, specifically to ßhCG hormone-secreting multinucleated syncytiotrophoblast and does not support induction of embryonic and extraembryonic lineages, extravillous trophoblast, and primitive endoderm. It has been previously reported that FGF2 can switch BMP4-induced hESC differentiation outcome to mesendoderm. Here, we show that FGF inhibition alone, or in combination with either ACTIVIN/NODAL inhibition or BMP activation, supports hESC differentiation to hCG-secreting syncytiotrophoblast. We show that the inhibition of the FGF pathway acts as a key in directing BMP4-mediated hESC differentiation to syncytiotrophoblast.

The origins of human embryonic stem cells: a biological conundrum.

Human inner cell mass (ICM) cells isolated from in vitro fertilized blastocysts are the progenitor cells used to establish in vitro stable human embryonic stem cells (hESCs) which are pluripotent and self-renew indefinitely. This long-term perpetuation of hESCs in the undifferentiated state is thought to be an in vitro adaptation of the ICM cells. To investigate at the molecular level how hESCs acquired their unique properties, transcriptional profiles of isolated ICM cells and undifferentiated hESCs were compared. We identified 33 genes enriched in the ICM compared to the trophectoderm and hESCs. These genes are involved in signaling cascades (SEMA7A and MAP3K10), cell proliferation (CUZD1 and MS4A7) and chromatin remodeling (H1FOO and HRMT1L4). Furthermore, primordial germ cell-specific genes (SGCA and TEX11) were detected as expressed in the ICM cells and not hESCs. We propose that the transcriptional differences observed between ICM cells and hESCs might be accounted for by adaptive reprogramming events induced by the in vitro culture conditions which are distinct from that of in vitro fertilized blastocysts. hESCs are a distinct cell type lacking in the human embryo but, nonetheless, resemble the ICM in their ability to differentiate into cells representative of the endodermal, ectodermal and mesodermal cell lineages.

Functional genomics of human pre-implantation development.

Early mammalian embryogenesis is currently the focus of intense interest because of the potential of inner cell mass-derived embryonic stem cell lines in new therapeutic strategies. As such, creating molecular profiles of gene expression during pre-implantation development will provide a framework for understanding the biological properties of these cells and also establish a tool set for subsequent functional studies. However, a major obstacle impeding progress in this area are moral issues regarding their use, the scarcity of these cells and the ability to successfully isolate and amplify enough mRNA from the minute amounts of total RNA present in these cells. The elucidation, unravelling and understanding the molecular basis of transcriptional control during pre-implantation development is of utmost importance if we are to diagnose, intervene, eliminate or reduce abnormalities associated with growth, disease and infertility by applying assisted reproduction. Importantly, these studies should enhance our knowledge of basic reproductive biology and its application to regenerative medicine. This review describes the application of in silico-based approaches, in order to obtain maximal information from published microarray-based gene expression data. For an illustration of this, we used gene expression data related to unfertilized oocytes and blastocysts to gain insights into genes and related signalling pathways (e.g. MAPK, PI3K, WNT, TGF-beta) involved in the switch from maternal to embryonic control of gene transcription during human pre-implantation development.

Conserved molecular portraits of bovine and human blastocysts as a consequence of the transition from maternal to embryonic control of gene expression.

The present study investigated mRNA expression profiles of bovine oocytes and blastocysts by using a cross-species hybridization approach employing an array consisting of 15,529 human cDNAs as probe, thus enabling the identification of conserved genes during human and bovine preimplantation development. Our analysis revealed 419 genes that were expressed in both oocytes and blastocysts. The expression of 1,324 genes was detected exclusively in the blastocyst, in contrast to 164 in the oocyte including a significant number of novel genes. Genes indicative for transcriptional and translational control (ELAVL4, TACC3) were overexpressed in the oocyte, whereas cellular trafficking (SLC2A14, SLC1A3), proteasome (PSMA1, PSMB3), cell cycle (BUB3, CCNE1, GSPT1), and protein modification and turnover (TNK1, UBE3A) genes were found to be overexpressed in blastocysts. Transcripts implicated in chromatin remodeling were found in both oocytes (NASP, SMARCA2) and blastocysts (H2AFY, HDAC7A). The trophectodermal markers PSG2 and KRT18 were enriched 5- and 50-fold in the blastocyst. Pathway analysis revealed differential expression of genes involved in 107 distinct signaling and metabolic pathways. For example, phosphatidylinositol signaling and gluconeogenesis were prominent pathways identified in the blastocyst. Expression patterns in bovine and human blastocysts were to a large extent identical. This analysis compared the transcriptomes of bovine oocytes and blastocysts and provides a solid foundation for future studies on the first major differentiation events in blastocysts and identification of a set of markers indicative for regular mammalian development.