Treating primary immunodeficiencies with defects in NK cells: from stem cell therapy to gene editing.

Primary immunodeficiency diseases (PIDs) are rare diseases that are characterized by genetic mutations that damage immunological function, defense, or both. Some of these rare diseases are caused by aberrations in the normal development of natural killer cells (NKs) or affect their lytic synapse. The pathogenesis of these types of diseases as well as the processes underlying target recognition by human NK cells is not well understood. Utilizing induced pluripotent stem cells (iPSCs) will aid in the study of human disorders, especially in the PIDs with defects in NK cells for PID disease modeling. This, together with genome editing technology, makes it possible for us to facilitate the discovery of future therapeutics and/or cell therapy treatments for these patients, because, to date, the only curative treatment available in the most severe cases is hematopoietic stem cell transplantation (HSCT). Recent progress in gene editing technology using CRISPR/Cas9 has significantly increased our capability to precisely modify target sites in the human genome. Among the many tools available for us to study human PIDs, disease- and patient-specific iPSCs together with gene editing offer unique and exceptional methodologies to gain deeper and more thorough understanding of these diseases as well as develop possible alternative treatment strategies. In this review, we will discuss some immunodeficiency disorders affecting NK cell function, such as classical NK deficiencies (CNKD), functional NK deficiencies (FNKD), and PIDs with involving NK cells as well as strategies to model and correct these diseases for further study and possible avenues for future therapies.

Generation of human organs in pigs via interspecies blastocyst complementation.

More than eighteen years have passed since the first derivation of human embryonic stem cells (ESCs), but their clinical use is still met with several challenges, such as ethical concerns regarding the need of human embryos, tissue rejection after transplantation and tumour formation. The generation of human induced pluripotent stem cells (iPSCs) enables the access to patient-derived pluripotent stem cells (PSCs) and opens the door for personalized medicine as tissues/organs can potentially be generated from the same genetic background as the patient recipients, thus avoiding immune rejections or complication of immunosuppression strategies. In this regard, successful replacement, or augmentation, of the function of damaged tissue by patient-derived differentiated stem cells provides a promising cell replacement therapy for many devastating human diseases. Although human iPSCs can proliferate unlimitedly in culture and harbour the potential to generate all cell types in the adult body, currently, the functionality of differentiated cells is limited. An alternative strategy to realize the full potential of human iPSC for regenerative medicine is the in vivo tissue generation in large animal species via interspecies blastocyst complementation. As this technology is still in its infancy and there remains more questions than answers, thus in this review, we mainly focus the discussion on the conceptual framework, the emerging technologies and recent advances involved with interspecies blastocyst complementation, and will refer the readers to other more in-depth reviews on dynamic pluripotent stem cell states, genome editing and interspecies chimeras. Likewise, other emerging alternatives to combat the growing shortage of human organs, such as xenotransplantation or tissue engineering, topics that has been extensively reviewed, will not be covered here.

Characterization of the Epigenetic Changes During Human Gonadal Primordial Germ Cells Reprogramming.

Epigenetic reprogramming is a central process during mammalian germline development. Genome-wide DNA demethylation in primordial germ cells (PGCs) is a prerequisite for the erasure of epigenetic memory, preventing the transmission of epimutations to the next generation. Apart from DNA demethylation, germline reprogramming has been shown to entail reprogramming of histone marks and chromatin remodelling. Contrary to other animal models, there is limited information about the epigenetic dynamics during early germ cell development in humans. Here, we provide further characterization of the epigenetic configuration of the early human gonadal PGCs. We show that early gonadal human PGCs are DNA hypomethylated and their chromatin is characterized by low H3K9me2 and high H3K27me3 marks. Similarly to previous observations in mice, human gonadal PGCs undergo dynamic chromatin changes concomitant with the erasure of genomic imprints. Interestingly, and contrary to mouse early germ cells, expression of BLIMP1/PRDM1 persists in through all gestational stages in human gonadal PGCs and is associated with nuclear lysine-specific demethylase-1. Our work provides important additional information regarding the chromatin changes associated with human PGCs development between 6 and 13 weeks of gestation in male and female gonads. Stem Cells 2016;34:2418-2428.

Olfactory stem cells reveal MOCOS as a new player in autism spectrum disorders.

With an onset under the age of 3 years, autism spectrum disorders (ASDs) are now understood as diseases arising from pre- and/or early postnatal brain developmental anomalies and/or early brain insults. To unveil the molecular mechanisms taking place during the misshaping of the developing brain, we chose to study cells that are representative of the very early stages of ontogenesis, namely stem cells. Here we report on MOlybdenum COfactor Sulfurase (MOCOS), an enzyme involved in purine metabolism, as a newly identified player in ASD. We found in adult nasal olfactory stem cells of 11 adults with ASD that MOCOS is downregulated in most of them when compared with 11 age- and gender-matched control adults without any neuropsychiatric disorders. Genetic approaches using in vivo and in vitro engineered models converge to indicate that altered expression of MOCOS results in neurotransmission and synaptic defects. Furthermore, we found that MOCOS misexpression induces increased oxidative-stress sensitivity. Our results demonstrate that altered MOCOS expression is likely to have an impact on neurodevelopment and neurotransmission, and may explain comorbid conditions, including gastrointestinal disorders. We anticipate our discovery to be a fresh starting point for the study on the roles of MOCOS in brain development and its functional implications in ASD clinical symptoms. Moreover, our study suggests the possible development of new diagnostic tests based on MOCOS expression, and paves the way for drug screening targeting MOCOS and/or the purine metabolism to ultimately develop novel treatments in ASD.

Activin A/BMP2 chimera AB235 drives efficient redifferentiation of long term cultured autologous chondrocytes.

Autologous chondrocyte implantation (ACI) depends on the quality and quantity of implanted cells and is hindered by the fact that chondrocytes cultured for long periods of time undergo dedifferentiation. Here we have developed a reproducible and efficient chondrogenic protocol to redifferentiate chondrocytes isolated from osteoarthritis (OA) patients. We used morphological, histological and immunological analysis together with a RT-PCR detection of collagen I and collagen II gene expression to show that chondrocytes isolated from articular cartilage biopsies of patients and subjected to long-term culture undergo dedifferentiation and that these cells can be redifferentiated following treatment with the chimeric Activin A/BMP2 ligand AB235. Examination of AB235-treated cell pellets in both in vitro and in vivo experiments revealed that redifferentiated chondrocytes synthesized a cartilage-specific extracellular matrix (ECM), primarily consisting of vertically-orientated collagen fibres and cartilage-specific proteoglycans. AB235-treated cell pellets also integrated into the surrounding subcutaneous tissue following transplantation in mice as demonstrated by their dramatic increase in size while non-treated control pellets disintegrated upon transplantation. Thus, our findings describe an effective protocol for the promotion of redifferentiation of autologous chondrocytes obtained from OA patients and the formation of a cartilage-like ECM that can integrate into the surrounding tissue in vivo.

Generation of iPSCs from genetically corrected Brca2 hypomorphic cells: implications in cell reprogramming and stem cell therapy.

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2(Δ) (27/) (Δ27)), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2(Δ) (27/) (Δ27) induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2(Δ) (27/) (Δ27) iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2(Δ) (27/) (Δ27) mouse embryonic fibroblasts. Gene-corrected Brca2(Δ) (27/) (Δ27) iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2(Δ) (27/) (Δ27) recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2(Δ) (27/) (Δ) (27) iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed.

Identification of phosphorylase kinase as a novel therapeutic target through high-throughput screening for anti-angiogenesis compounds in zebrafish.

Angiogenesis is essential for development and tumor progression. With the aim of identifying new compound inhibitors of the angiogenesis process, we used an established enhanced green fluorescent protein-transgenic zebrafish line to develop an automated assay that enables high-throughput screening of compound libraries in a whole-organism setting. Using this system, we have identified novel kinase inhibitor compounds that show anti-angiogenic properties in both zebrafish in-vivo system and in human endothelial cell in-vitro angiogenesis models. Furthermore, we have determined the kinase target of these compounds and have identified and validated a previously uncharacterized involvement of phosphorylase kinase subunit G1 (PhKG1) in angiogenesis in vivo. In addition, we have found that PhKG1 is upregulated in human tumor samples and that aberrations in gene copy number of PhK subunits are a common feature of human tumors. Our results provide a novel insight into the angiogenesis process, as well as identify new potential targets for anti-angiogenic therapies.

Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and induced pluripotent stem cells.

Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) offer great hope for in vitro modeling of Parkinson's disease (PD), as well as for designing cell-replacement therapies. To realize these opportunities, there is an urgent need to develop efficient protocols for the directed differentiation of hESC/iPSC into dopamine (DA) neurons with the specific characteristics of the cell population lost to PD, i.e., A9-subtype ventral midbrain DA neurons. Here we use lentiviral vectors to drive the expression of LMX1A, which encodes a transcription factor critical for ventral midbrain identity, specifically in neural progenitor cells. We show that clonal lines of hESC engineered to contain one or two copies of this lentiviral vector retain long-term self-renewing ability and pluripotent differentiation capacity. Greater than 60% of all neurons generated from LMX1A-engineered hESC were ventral midbrain DA neurons of the A9 subtype, compared with ∼10% in green fluorescent protein-engineered controls, as judged by specific marker expression and functional analyses. Moreover, DA neuron precursors differentiated from LMX1A-engineered hESC were able to survive and differentiate when grafted into the brain of adult mice. Finally, we provide evidence that LMX1A overexpression similarly increases the yield of DA neuron differentiation from human iPSC. Taken together, our data show that stable genetic engineering of hESC/iPSC with lentiviral vectors driving controlled expression of LMX1A is an efficient way to generate enriched populations of human A9-subtype ventral midbrain DA neurons, which should prove useful for modeling PD and may be helpful for designing future cell-replacement strategies.

Ablation of Dido3 compromises lineage commitment of stem cells in vitro and during early embryonic development.

The death inducer obliterator (Dido) locus encodes three protein isoforms, of which Dido3 is the largest and most broadly expressed. Dido3 is a nuclear protein that forms part of the spindle assembly checkpoint (SAC) and is necessary for correct chromosome segregation in somatic and germ cells. Here we report that specific ablation of Dido3 function in mice causes lethal developmental defects at the onset of gastrulation. Although these defects are associated with centrosome amplification, spindle malformation and a DNA damage response, we provide evidence that embryonic lethality of the Dido3 mutation cannot be explained by its impact on chromosome segregation alone. We show that loss of Dido3 expression compromises differentiation of embryonic stem cells in vitro and of epiblast cells in vivo, resulting in early embryonic death at around day 8.5 of gestation. Close analysis of Dido3 mutant embryoid bodies indicates that ablation of Dido3, rather than producing a generalized differentiation blockade, delays the onset of lineage commitment at the primitive endoderm specification stage. The dual role of Dido3 in chromosome segregation and stem cell differentiation supports the implication of SAC components in stem cell fate decisions.

Complete meiosis from human induced pluripotent stem cells.

Gamete failure-derived infertility affects millions of people worldwide; for many patients, gamete donation by unrelated donors is the only available treatment. Embryonic stem cells (ESCs) can differentiate in vitro into germ-like cells, but they are genetically unrelated to the patient. Using an in vitro protocol that aims at recapitulating development, we have achieved, for the first time, complete differentiation of human induced pluripotent stem cells (hiPSCs) to postmeiotic cells. Unlike previous reports using human ESCs, postmeiotic cells arose without the over-expression of germline related transcription factors. Moreover, we consistently obtained haploid cells from hiPSCs of different origin (keratinocytes and cord blood), produced with a different number of transcription factors, and of both genetic sexes, suggesting the independence of our approach from the epigenetic memory of the reprogrammed somatic cells. Our work brings us closer to the production of personalized human gametes in vitro.

Generation of cardiomyocytes from new human embryonic stem cell lines derived from poor-quality blastocysts.

Human embryonic stem (hES) cells represent a potential source for cell replacement therapy of many degenerative diseases. Most frequently, hES cell lines are derived from surplus embryos from assisted reproduction cycles, independent of their quality or morphology. Here, we show that hES cell lines can be obtained from poor-quality blastocysts with the same efficiency as that obtained from good- or intermediate-quality blastocysts. Furthermore, we show that the self-renewal, pluripotency, and differentiation ability of hES cell lines derived from either source are comparable. Finally, we present a simple and reproducible embryoid body-based protocol for the differentiation of hES cells into functional cardiomyocytes. The five new hES cell lines derived here should widen the spectrum of available resources for investigating the biology of hES cells and advancing toward efficient strategies of regenerative medicine.

Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse.

Dickkopf1 (Dkk1) is a secreted protein that acts as a Wnt inhibitor and, together with BMP inhibitors, is able to induce the formation of ectopic heads in Xenopus. Here, we show that Dkk1 null mutant embryos lack head structures anterior of the midbrain. Analysis of chimeric embryos implicates the requirement of Dkk1 in anterior axial mesendoderm but not in anterior visceral endoderm for head induction. In addition, mutant embryos show duplications and fusions of limb digits. Characterization of the limb phenotype strongly suggests a role for Dkk1 both in cell proliferation and in programmed cell death. Our data provide direct genetic evidence for the requirement of secreted Wnt antagonists during embryonic patterning and implicate Dkk1 as an essential inducer during anterior specification as well as a regulator during distal limb patterning.

Patterning mechanisms controlling vertebrate limb development.

Vertebrate limb buds are embryonic structures for which much molecular and cellular data are known regarding the mechanisms that control pattern formation during development. Specialized regions of the developing limb bud, such as the zone of polarizing activity (ZPA), the apical ectodermal ridge (AER), and the non-ridge ectoderm, direct and coordinate the development of the limb bud along the anterior-posterior (AP), dorsal-ventral (DV), and proximal-distal (PD) axes, giving rise to a stereotyped pattern of elements well conserved among tetrapods. In recent years, specific gene functions have been shown to mediate the organizing and patterning activities of the ZPA, the AER, and the non-ridge ectoderm. The analysis of these gene functions has revealed the existence of complex interactions between signaling pathways operated by secreted factors of the HH, TGF-beta/BMP, WNT, and FGF superfamilies, which interact with many other genetic networks to control limb positioning, outgrowth, and patterning. The study of limb development has helped to establish paradigms for the analysis of pattern formation in many other embryonic structures and organs.

Wnt signaling and PKA control Nodal expression and left-right determination in the chick embryo.

Expression of the Nodal gene, which encodes a member of the TGFbeta superfamily of secreted factors, localizes to the left side of the developing embryo in all vertebrates examined so far. This asymmetric pattern correlates with normal development of the left-right axis. We now show that the Wnt and PKA signaling pathways control left-right determination in the chick embryo through Nodal. A Wnt/beta-catenin pathway controls Nodal expression in and around Hensen's node, without affecting the upstream regulators Sonic hedgehog, Car and Fibroblast Growth Factor 8. Transcription of Nodal is also positively regulated by a protein kinase A-dependent pathway. Both the adhesion protein N-cadherin and PKI (an endogenous protein kinase A inhibitor) are localized to the right side of the node and may contribute to restrict Nodal activation by Wnt signaling and PKA to the left side of the node.

WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo.

A regulatory loop between the fibroblast growth factors FGF-8 and FGF-10 plays a key role in limb initiation and AER induction in vertebrate embryos. Here, we show that three WNT factors signaling through beta-catenin act as key regulators of the FGF-8/FGF-10 loop. The Wnt-2b gene is expressed in the intermediate mesoderm and the lateral plate mesoderm in the presumptive chick forelimb region. Cells expressing Wnt-2b are able to induce Fgf-10 and generate an extra limb when implanted into the flank. In the presumptive hindlimb region, another Wnt gene, Wnt-8c, controls Fgf-10 expression, and is also capable of inducing ectopic limb formation in the flank. Finally, we also show that the induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-3a. Thus, three WNT signals mediated by beta-catenin control both limb initiation and AER induction in the vertebrate embryo.

Development of the limb neuromuscular system.

Appendages, such as wings of a fly or limbs of a vertebrate, are excellent models to study the principles of patterning and morphogenesis. In the adult these structures are used for a variety of behaviors, including locomotion. Although support structures of the adult vertebrate limb are generated within the limb bud, its dynamic elements are derived from the somitic mesoderm and neural tube. Recent studies show that regional patterns set up in the mesenchyme-filled limb bud guide muscle precursors and developing motor axons to their proper location within the limb. Subsequent development of the neuromuscular system is regulated by cell surface interactions between pre-specified muscle fibers and motor axons.

Asymmetric nodal signaling in the zebrafish diencephalon positions the pineal organ.

The vertebrate brain develops from a bilaterally symmetric neural tube but later displays profound anatomical and functional asymmetries. Despite considerable progress in deciphering mechanisms of visceral organ laterality, the genetic pathways regulating brain asymmetries are unknown. In zebrafish, genes implicated in laterality of the viscera (cyclops/nodal, antivin/lefty and pitx2) are coexpressed on the left side of the embryonic dorsal diencephalon, within a region corresponding to the presumptive epiphysis or pineal organ. Asymmetric gene expression in the brain requires an intact midline and Nodal-related factors. RNA-mediated rescue of mutants defective in Nodal signaling corrects tissue patterning at gastrulation, but fails to restore left-sided gene expression in the diencephalon. Such embryos develop into viable adults with seemingly normal brain morphology. However, the pineal organ, which typically emanates at a left-to-medial site from the dorsal diencephalic roof, becomes displaced in position. Thus, a conserved signaling pathway regulating visceral laterality also underlies an anatomical asymmetry of the zebrafish forebrain.

Evidence that members of the Cut/Cux/CDP family may be involved in AER positioning and polarizing activity during chick limb development.

In vertebrates, the apical ectodermal ridge (AER) is a specialized epithelium localized at the dorsoventral boundary of the limb bud that regulates limb outgrowth. In Drosophila, the wing margin is also a specialized region located at the dorsoventral frontier of the wing imaginal disc. The wingless and Notch pathways have been implicated in positioning both the wing margin and the AER. One of the nuclear effectors of the Notch signal in the wing margin is the transcription factor cut. Here we report the identification of two chick homologues of the Cut/Cux/CDP family that are expressed in the developing limb bud. Chick cux1 is expressed in the ectoderm outside the AER, as well as around ridge-like structures induced by (&bgr;)-catenin, a downstream target of the Wnt pathway. cux1 overexpression in the chick limb results in scalloping of the AER and limb truncations, suggesting that Cux1 may have a role in limiting the position of the AER by preventing the ectodermal cells around it from differentiating into AER cells. The second molecule of the Cut family identified in this study, cux2, is expressed in the pre-limb lateral plate mesoderm, posterior limb bud and flank mesenchyme, a pattern reminiscent of the distribution of polarizing activity. The polarizing activity is determined by the ability of a certain region to induce digit duplications when grafted into the anterior margin of a host limb bud. Several manipulations of the chick limb bud show that cux2 expression is regulated by retinoic acid, Sonic hedgehog and the posterior AER. These results suggest that Cux2 may have a role in generating or mediating polarizing activity. Taking into account the probable involvement of Cut/Cux/CDP molecules in cell cycle regulation and differentiation, our results raise the hypothesis that chick Cux1 and Cux2 may act by modulating proliferation versus differentiation in the limb ectoderm and polarizing activity regions, respectively.