Lentivirus-mediated gene therapy corrects ribosomal biogenesis and shows promise for Diamond Blackfan anemia.

This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral vector, PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK) already used in different gene therapy trials, including FA gene therapy. In the latter one, EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells, providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was verified using a potentially novel method applicable to patients' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.

Effect of virgin olive oil as spreadable preparation on atherosclerosis compared to dairy butter in Apoe-deficient mice.

Olive oil is the main source of lipid energy in the Mediterranean diet and there is strong evidence of its health benefits. The effect of extra virgin olive oil (EVOO) in the form of a preparation of spreadable virgin olive oil (S-VO) on the progression of atheroma plaques was investigated in Apoe-deficient mice, a model of accelerated atherosclerosis. METHODS: Two isocaloric Western purified diets containing 20% fat, either as S-VO or as dairy butter, were used to feed 28 males and 16 females of two-month-old Apoe-deficient mice for 12 weeks. S-VO was prepared by blending more than 75% virgin olive oil with other vegetal natural fat to obtain a solid fat. Plasma total cholesterol, triglycerides and HDL cholesterol were measured. Hepatic lipid droplets were analyzed. Areas of atherosclerotic aortic lesions were quantified in cross-sectional images of the proximal aorta and en face analysis of the whole aorta. RESULTS: Total plasma cholesterol was increased in mice on the butter-supplemented diet in both female and male mice compared to S-VO, and the ratio of TC/HDL-cholesterol was significantly lower in S-VO than in the butter diet, although only in males, and no differences in plasma triglycerides were observed. No significant differences in hepatic lipid droplets were observed between diets in either sex. Aortic lesion areas were significantly higher in mice consuming the butter versus the S-VO diet in both sexes. CONCLUSION: Extra virgin olive oil prepared in spreadable form maintained the delay in atheroma plaque progression compared to butter.

Restored glyoxylate metabolism after AGXT gene correction and direct reprogramming of primary hyperoxaluria type 1 fibroblasts.

Primary hyperoxaluria type 1 (PH1) is a rare inherited metabolic disorder characterized by oxalate overproduction in the liver, resulting in renal damage. It is caused by mutations in the AGXT gene. Combined liver and kidney transplantation is currently the only permanent curative treatment. We combined locus-specific gene correction and hepatic direct cell reprogramming to generate autologous healthy induced hepatocytes (iHeps) from PH1 patient-derived fibroblasts. First, site-specific AGXT corrected cells were obtained by homology directed repair (HDR) assisted by CRISPR-Cas9, following two different strategies: accurate point mutation (c.731T>C) correction or knockin of an enhanced version of AGXT cDNA. Then, iHeps were generated, by overexpression of hepatic transcription factors. Generated AGXT-corrected iHeps showed hepatic gene expression profile and exhibited in vitro reversion of oxalate accumulation compared to non-edited PH1-derived iHeps. This strategy set up a potential alternative cellular source for liver cell replacement therapy and a personalized PH1 in vitro disease model.

Soluble mutant huntingtin drives early human pathogenesis in Huntington's disease.

Huntington's disease (HD) is an incurable inherited brain disorder characterised by massive degeneration of striatal neurons, which correlates with abnormal accumulation of misfolded mutant huntingtin (mHTT) protein. Research on HD has been hampered by the inability to study early dysfunction and progressive degeneration of human striatal neurons in vivo. To investigate human pathogenesis in a physiologically relevant context, we transplanted human pluripotent stem cell-derived neural progenitor cells (hNPCs) from control and HD patients into the striatum of new-born mice. Most hNPCs differentiated into striatal neurons that projected to their target areas and established synaptic connexions within the host basal ganglia circuitry. Remarkably, HD human striatal neurons first developed soluble forms of mHTT, which primarily targeted endoplasmic reticulum, mitochondria and nuclear membrane to cause structural alterations. Furthermore, HD human cells secreted extracellular vesicles containing mHTT monomers and oligomers, which were internalised by non-mutated mouse striatal neurons triggering cell death. We conclude that interaction of mHTT soluble forms with key cellular organelles initially drives disease progression in HD patients and their transmission through exosomes contributes to spread the disease in a non-cell autonomous manner.

Specific correction of pyruvate kinase deficiency-causing point mutations by CRISPR/Cas9 and single-stranded oligodeoxynucleotides.

Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene. PKD-erythroid cells suffer from an energy imbalance caused by a reduction of erythroid pyruvate kinase (RPK) enzyme activity. PKD is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. More than 300 disease-causing mutations have been identified as causing PKD. Most mutations are missense mutations, commonly present as compound heterozygous. Therefore, specific correction of these point mutations might be a promising therapy for the treatment of PKD patients. We have explored the potential of precise gene editing for the correction of different PKD-causing mutations, using a combination of single-stranded oligodeoxynucleotides (ssODN) with the CRISPR/Cas9 system. We have designed guide RNAs (gRNAs) and single-strand donor templates to target four different PKD-causing mutations in immortalized patient-derived lymphoblastic cell lines, and we have detected the precise correction in three of these mutations. The frequency of the precise gene editing is variable, while the presence of additional insertions/deletions (InDels) has also been detected. Significantly, we have identified high mutation-specificity for two of the PKD-causing mutations. Our results demonstrate the feasibility of a highly personalized gene-editing therapy to treat point mutations in cells derived from PKD patients.

Lack of EGFR catalytic activity in hepatocytes improves liver regeneration following DDC-induced cholestatic injury by promoting a pro-restorative inflammatory response.

Despite the well-known hepatoprotective role of the epidermal growth factor receptor (EGFR) pathway upon acute damage, its specific actions during chronic liver disease, particularly cholestatic injury, remain ambiguous and unresolved. Here, we analyzed the consequences of inactivating EGFR signaling in the liver on the regenerative response following cholestatic injury. For that, transgenic mice overexpressing a dominant negative mutant human EGFR lacking tyrosine kinase activity (ΔEGFR) in albumin-positive cells were submitted to liver damage induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an experimental model resembling human primary sclerosing cholangitis. Our results show an early activation of EGFR after 1-2 days of a DDC-supplemented diet, followed by a signaling switch-off. Furthermore, ΔEGFR mice showed less liver damage and a more efficient regeneration following DDC injury. Analysis of the mechanisms driving this effect revealed an enhanced activation of mitogenic/survival signals, AKT and ERK1/2-MAPKs, and changes in cell turnover consistent with a quicker resolution of damage in response to DDC. These changes were concomitant with profound differences in the profile of intrahepatic immune cells, consisting of a shift in the M1/M2 balance towards M2 polarity, and the Cd4/Cd8 ratio in favor of Cd4 lymphocytes, overall supporting an immune cell switch into a pro-restorative phenotype. Interestingly, ΔEGFR livers also displayed an amplified ductular reaction, with increased expression of EPCAM and an increased number of CK19-positive ductular structures in portal areas, demonstrating an overexpansion of ductular progenitor cells. In summary, our work supports the notion that hepatocyte-specific EGFR activity acts as a key player in the crosstalk between parenchymal and non-parenchymal hepatic cells, promoting the pro-inflammatory response activated during cholestatic injury and therefore contributing to the pathogenesis of cholestatic liver disease. © 2022 The Pathological Society of Great Britain and Ireland.

Efficient and Fast Generation of Relevant Disease Mouse Models by In Vitro and In Vivo Gene Editing of Zygotes.

Knockout mice for human disease-causing genes provide valuable models in which new therapeutic approaches can be tested. Electroporation of genome editing tools into zygotes, in vitro or within oviducts, allows for the generation of targeted mutations in a shorter time. We have generated mouse models deficient in genes involved in metabolic rare diseases (Primary Hyperoxaluria Type 1 Pyruvate Kinase Deficiency) or in a tumor suppressor gene (Rasa1). Pairs of guide RNAs were designed to generate controlled deletions that led to the absence of protein. In vitro or in vivo ribonucleoprotein (RNP) electroporation rendered more than 90% and 30% edited newborn animals, respectively. Mice lines with edited alleles were established and disease hallmarks have been verified in the three models that showed a high consistency of results and validating RNP electroporation into zygotes as an efficient technique for disease modeling without the need to outsource to external facilities.

Upregulation of NKG2D ligands impairs hematopoietic stem cell function in Fanconi anemia.

Fanconi anemia (FA) is the most prevalent inherited bone marrow failure (BMF) syndrome. Nevertheless, the pathophysiological mechanisms of BMF in FA have not been fully elucidated. Since FA cells are defective in DNA repair, we hypothesized that FA hematopoietic stem and progenitor cells (HSPCs) might express DNA damage-associated stress molecules such as natural killer group 2 member D ligands (NKG2D-Ls). These ligands could then interact with the activating NKG2D receptor expressed in cytotoxic NK or CD8+ T cells, which may result in progressive HSPC depletion. Our results indeed demonstrated upregulated levels of NKG2D-Ls in cultured FA fibroblasts and T cells, and these levels were further exacerbated by mitomycin C or formaldehyde. Notably, a high proportion of BM CD34+ HSPCs from patients with FA also expressed increased levels of NKG2D-Ls, which correlated inversely with the percentage of CD34+ cells in BM. Remarkably, the reduced clonogenic potential characteristic of FA HSPCs was improved by blocking NKG2D-NKG2D-L interactions. Moreover, the in vivo blockage of these interactions in a BMF FA mouse model ameliorated the anemia in these animals. Our study demonstrates the involvement of NKG2D-NKG2D-L interactions in FA HSPC functionality, suggesting an unexpected role of the immune system in the progressive BMF that is characteristic of FA.

Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations.

Factor V deficiency, an ultra-rare congenital coagulopathy, is characterized by bleeding episodes that may be more or less intense as a function of the levels of coagulation factor activity present in plasma. Fresh-frozen plasma, often used to treat patients with factor V deficiency, is a scarcely effective palliative therapy with no specificity to the disease. CRISPR/Cas9-mediated gene editing, following precise deletion by non-homologous end-joining, has proven to be highly effective for modeling on a HepG2 cell line a mutation similar to the one detected in the factor V-deficient patient analyzed in this study, thus simulating the pathological phenotype. Additional CRISPR/Cas9-driven non-homologous end-joining precision deletion steps allowed correction of 41% of the factor V gene mutated cells, giving rise to a newly developed functional protein. Taking into account the plasma concentrations corresponding to the different levels of severity of factor V deficiency, it may be argued that the correction achieved in this study could, in ideal conditions, be sufficient to turn a severe phenotype into a mild or asymptomatic one.

Gene Editing for Inherited Red Blood Cell Diseases.

Today gene therapy is a real therapeutic option to address inherited hematological diseases that could be beneficial for thousands of patients worldwide. Currently, gene therapy is used to treat different monogenic hematological pathologies, including several red blood cell diseases such as ß-thalassemia, sickle cell disease and pyruvate kinase deficiency. This approach is based on addition gene therapy, which consists of the correction of hematopoietic stem cells (HSCs) using lentiviral vectors, which integrate a corrected version of the altered gene. Lentivirally-corrected HSCs generate healthy cells that compensate for the deficiency caused by genetic mutations. Despite its successful results, this approach lacks both control of the integration of the transgene into the genome and endogenous regulation of the therapeutic gene, both of which are important aspects that might be a cause for concern. To overcome these limitations, gene editing is able to correct the altered gene through more precise and safer approaches. Cheap and easy-to-design gene editing tools, such as the CRISPR/Cas9 system, allow the specific correction of the altered gene without affecting the rest of the genome. Inherited erythroid diseases, such as thalassemia, sickle cell disease and Pyruvate Kinase Deficiency, have been the test bed for these gene editing strategies, and promising results are currently being seen. CRISPR/Cas9 system has been successfully used to manipulate globin regulation to re-activate fetal globin chains in adult red blood cells and to compensate for hemoglobin defects. Knock-in at the mutated locus to express the therapeutic gene under the endogenous gene regulatory region has also been accomplished successfully. Thanks to the lessons learned from previous lentiviral gene therapy research and trials, gene editing for red blood cell diseases is rapidly moving from its proof-of-concept to its first exciting results in the clinic. Indeed, patients suffering from ß-thalassemia and sickle cell disease have already been successfully treated with gene editing, which will hopefully inspire the use of gene editing to cure erythroid disorders and many other inherited diseases in the near future.

Clinically relevant gene editing in hematopoietic stem cells for the treatment of pyruvate kinase deficiency.

Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell (P KLR) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients.

Preclinical studies of efficacy thresholds and tolerability of a clinically ready lentiviral vector for pyruvate kinase deficiency treatment.

Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disorder caused by mutations in the PKLR gene. PKD is characterized by non-spherocytic hemolytic anemia of variable severity and may be fatal in some cases during early childhood. Although not considered the standard of care, allogeneic stem cell transplantation has been shown as a potentially curative treatment, limited by donor availability, toxicity, and incomplete engraftment. Preclinical studies were conducted to define conditions to enable consistent therapeutic reversal, which were based on our previous data on lentiviral gene therapy for PKD. Improvement of erythroid parameters was identified by the presence of 20%-30% healthy donor cells. A minimum vector copy number (VCN) of 0.2-0.3 was required to correct PKD when corrected cells were transplanted in a mouse model for PKD. Biodistribution and pharmacokinetics studies, with the aim of conducting a global gene therapy clinical trial for PKD patients (RP-L301-0119), demonstrated that genetically corrected cells do not confer additional side effects. Moreover, a clinically compatible transduction protocol with mobilized peripheral blood CD34+ cells was optimized, thus facilitating the efficient transduction on human cells capable of repopulating the hematopoiesis of immunodeficient mice. We established conditions for a curative lentiviral vector gene therapy protocol for PKD.

Enhanced anti-inflammatory effects of mesenchymal stromal cells mediated by the transient ectopic expression of CXCR4 and IL10.

BACKGROUND: Mesenchymal stromal cells (MSCs) constitute one of the cell types most frequently used in cell therapy. Although several studies have shown the efficacy of these cells to modulate inflammation in different animal models, the results obtained in human clinical trials have been more modest. Here, we aimed at improving the therapeutic properties of MSCs by inducing a transient expression of two molecules that could enhance two different properties of these cells. With the purpose of improving MSC migration towards inflamed sites, we induced a transient expression of the C-X-C chemokine receptor type 4 (CXCR4). Additionally, to augment the anti-inflammatory properties of MSCs, a transient expression of the anti-inflammatory cytokine, interleukin 10 (IL10), was also induced. METHODS: Human adipose tissue-derived MSCs were transfected with messenger RNAs carrying the codon-optimized versions of CXCR4 and/or IL10. mRNA-transfected MSCs were then studied, first to evaluate whether the characteristic phenotype of MSCs was modified. Additionally, in vitro and also in vivo studies in an LPS-induced inflamed pad model were conducted to evaluate the impact associated to the transient expression of CXCR4 and/or IL10 in MSCs. RESULTS: Transfection of MSCs with CXCR4 and/or IL10 mRNAs induced a transient expression of these molecules without modifying the characteristic phenotype of MSCs. In vitro studies then revealed that the ectopic expression of CXCR4 significantly enhanced the migration of MSCs towards SDF-1, while an increased immunosuppression was associated with the ectopic expression of IL10. Finally, in vivo experiments showed that the co-expression of CXCR4 and IL10 increased the homing of MSCs into inflamed pads and induced an enhanced anti-inflammatory effect, compared to wild-type MSCs. CONCLUSIONS: Our results demonstrate that the transient co-expression of CXCR4 and IL10 enhances the therapeutic potential of MSCs in a local inflammation mouse model, suggesting that these mRNA-modified cells may constitute a new step in the development of more efficient cell therapies for the treatment of inflammatory diseases.

Natural estrogens enhance the engraftment of human hematopoietic stem and progenitor cells in immunodeficient mice.

Hematopoietic Stem and Progenitor Cells are crucial in the maintenance of lifelong production of all blood cells. These Stem Cells are highly regulated to maintain homeostasis through a delicate balance between quiescence, self-renewal and differentiation. However, this balance is altered during the hematopoietic recovery after Hematopoietic Stem and Progenitor Cell Transplantation. Transplantation efficacy can be limited by inadequate Hematopoietic Stem Cells number, poor homing, low level of engraftment, or limited self-renewal. As recent evidences indicate that estrogens are involved in regulating the hematopoiesis, we sought to examine whether natural estrogens (estrone or E1, estradiol or E2, estriol or E3 and estetrol or E4) modulate human Hematopoietic Stem and Progenitor Cells. Our results show that human Hematopoietic Stem and Progenitor Cell subsets express estrogen receptors, and whose signaling is activated by E2 and E4 on these cells. Additionally, these natural estrogens cause different effects on human Progenitors in vitro. We found that both E2 and E4 expand human Hematopoietic Stem and Progenitor Cells. However, E4 was the best tolerated estrogen and promoted cell cycle of human Hematopoietic Progenitors. Furthermore, we identified that E2 and, more significantly, E4 doubled human hematopoietic engraftment in immunodeficient mice without altering other Hematopoietic Stem and Progenitor Cells properties. Finally, the impact of E4 on promoting human hematopoietic engraftment in immunodeficient mice might be mediated through the regulation of mesenchymal stromal cells in the bone marrow niche. Together, our data demonstrate that E4 is well tolerated and enhances human reconstitution in immunodeficient mice, directly by modulating human Hematopoietic Progenitor properties and indirectly by interacting with the bone marrow niche. This application might have particular relevance to ameliorate the hematopoietic recovery after myeloablative conditioning, especially when limiting numbers of Hematopoietic Stem and Progenitor Cells are available.

Generation of NKX2.5GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts.

Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5GFP reporter cardiac fibroblasts. We first established a new NKX2.5GFP reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5GFP hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5GFP cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.

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.

Human Pluripotent Stem Cell-Derived Neurons Are Functionally Mature In Vitro and Integrate into the Mouse Striatum Following Transplantation.

Human pluripotent stem cells (hPSCs) are a powerful tool for modelling human development. In recent years, hPSCs have become central in cell-based therapies for neurodegenerative diseases given their potential to replace affected neurons. However, directing hPSCs into specific neuronal types is complex and requires an accurate protocol that mimics endogenous neuronal development. Here we describe step-by-step a fast feeder-free neuronal differentiation protocol to direct hPSCs to mature forebrain neurons in 37 days in vitro (DIV). The protocol is based upon a combination of specific morphogens, trophic and growth factors, ions, neurotransmitters and extracellular matrix elements. A human-induced PSC line (Ctr-Q33) and a human embryonic stem cell line (GEN-Q18) were used to reinforce the potential of the protocol. Neuronal activity was analysed by single-cell calcium imaging. At 8 DIV, we obtained a homogeneous population of hPSC-derived neuroectodermal progenitors which self-arranged in bi-dimensional neural tube-like structures. At 16 DIV, we generated hPSC-derived neural progenitor cells (NPCs) with mostly a subpallial identity along with a subpopulation of pallial NPCs. Terminal in vitro neuronal differentiation was confirmed by the expression of microtubule associated protein 2b (Map 2b) by almost 100% of hPSC-derived neurons and the expression of specific-striatal neuronal markers including GABA, CTIP2 and DARPP-32. HPSC-derived neurons showed mature and functional phenotypes as they expressed synaptic markers, voltage-gated ion channels and neurotransmitter receptors. Neurons displayed diverse spontaneous activity patterns that were classified into three major groups, namely "high", "intermediate" and "low" firing neurons. Finally, transplantation experiments showed that the NPCs survived and differentiated within mouse striatum for at least 3 months. NPCs integrated host environmental cues and differentiated into striatal medium-sized spiny neurons (MSNs), which successfully integrated into the endogenous circuitry without teratoma formation. Altogether, these findings demonstrate the potential of this robust human neuronal differentiation protocol, which will bring new opportunities for the study of human neurodevelopment and neurodegeneration, and will open new avenues in cell-based therapies, pharmacological studies and alternative in vitro toxicology.

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 ß2 integrins, which are expressed on hematopoietic cells. ß2 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.