Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells

Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.info:eu-repo/semantics/publishedVersio

Dopamine D1 Receptors Regulate Spines in Striatal Direct-Pathway and Indirect-Pathway Neurons

Background: Dopamine transmission is involved in the maintenance of the structural plasticity of direct-pathway and indirect-pathway striatal projection neurons (d-SPNs and i-SPNs, respectively). The lack of dopamine in Parkinson's disease produces synaptic remodeling in both types of SPNs, reducing the length of the dendritic arbor and spine density and increasing the intrinsic excitability. Meanwhile, the elevation of dopamine levels by levodopa recovers these alterations selectively in i-SPNs. However, little is known about the specific role of the D1 receptor (D1R) in these alterations. Methods: To explore the specific role of D1R in the synaptic remodeling of SPNs, we used knockout D1R mice (D1R) and wild-type mice crossed with drd2-enhanced green fluorescent protein (eGFP) to identify d-SPNs and i-SPNs. Corticostriatal slices were used for reconstruction of the dendritic arbors after Lucifer yellow intracellular injection and for whole-cell recordings in naïve and parkinsonian mice treated with saline or levodopa. Results: The genetic inactivation of D1R reduces the length of the dendritic tree and the spine density in all SPNs, although more so in d-SPNs, which also increases their spiking. In parkinsonian D1R mice, the spine density decreases in i-SPNs, and this spine loss recovers after chronic levodopa. Conclusions: D1R is essential for the maintenance of spine plasticity in d-SPNs but also affects i-SPNs, indicating an important crosstalk between these 2 types of neurons. © 2020 International Parkinson and Movement Disorder Society.This work was supported by grants from the Spanish Ministries of Science and Innovation (SAF2016‐78207‐R and PCIN‐2015‐098) and of Sanidad Consumo y Bienestar Social, ISCIII, CIBERNED CB06/05/0055, PNSD2016I033) and 172275 from Ramón Areces Foundation to RM. AS has a predoctoral fellow of Comunidad de Madrid

Dopamine D2R is Required for Hippocampal-dependent Memory and Plasticity at the CA3-CA1 Synapse

Dopamine receptors play an important role in motivational, emotional, and motor responses. In addition, growing evidence suggests a key role of hippocampal dopamine receptors in learning and memory. It is well known that associative learning and synaptic plasticity of CA3-CA1 requires the dopamine D1 receptor (D1R). However, the specific role of the dopamine D2 receptor (D2R) on memory-related neuroplasticity processes is still undefined. Here, by using two models of D2R loss, D2R knockout mice (Drd2-/-) and mice with intrahippocampal injections of Drd2-small interfering RNA (Drd2-siRNA), we aimed to investigate how D2R is involved in learning and memory as well as in long-term potentiation of the hippocampus. Our studies revealed that the genetic inactivation of D2R impaired the spatial memory, associative learning, and the classical conditioning of eyelid responses. Similarly, deletion of D2R reduced the activity-dependent synaptic plasticity in the hippocampal CA1-CA3 synapse. Our results demonstrate the first direct evidence that D2R is essential in behaving mice for trace eye blink conditioning and associated changes in hippocampal synaptic strength. Taken together, these results indicate a key role of D2R in regulating hippocampal plasticity changes and, in consequence, acquisition and consolidation of spatial and associative forms of memory.Spanish Ministries of Science and Innovation (PID2019-111693RBI00, PCIN-2015-098); Ramón Areces Foundation (ref 172275); European Union’s Horizon 2020 research and innovation program, AND-PD (grant agreement no. 848002 to R.M., BFU2017- 82375-R to A.G. and J.M.D.-G.); Health, Social Services and Equality (PI2019/09-3), CIBERNED CB06/05/0055 to R.M.); the Spanish Junta de Andalucía (BIO-122, PY18FR_823 to A.G. and J.M.D.-G.)

The downregulated membrane expression of CD18 in CD34+ cells defines a primitive population of human hematopoietic stem cells.

BACKGROUND: CD18 is the common beta subunit of β2 integrins, which are expressed on hematopoietic cells. β2 integrins are essential for cell adhesion and leukocyte trafficking. METHODS: Here we have analyzed the expression of CD18 in different subsets of human hematopoietic stem and progenitor cells (HSPCs) from cord blood (CB), bone marrow (BM), and mobilized peripheral blood (mPB) samples. CD34+ cells were classified into CD18high and CD18low/neg, and each of these populations was analyzed for the expression of HSPC markers, as well as for their clonogenity, quiescence state, and repopulating ability in immunodeficient mice. RESULTS: A downregulated membrane expression of CD18 was associated with a primitive hematopoietic stem cells (HSC) phenotype, as well as with a higher content of quiescent cells and multipotent colony-forming cells (CFCs). Although no differences in the short-term repopulating potential of CD18low/neg CD34+ and CD18high CD34+ cells were observed, CD18low/neg CD34+ cells were characterized by an enhanced long-term repopulating ability in NSG mice. CONCLUSIONS: Overall, our results indicate that the downregulated membrane expression of CD18 characterizes a primitive population of human hematopoietic repopulating cells

In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9

The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30–60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated

In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9

The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30–60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated