The Role of Rab GTPases in the development of genetic and malignant diseases.

Small GTPases have been shown to play an important role in several cellular functions, including cytoskeletal remodeling, cell polarity, intracellular trafficking, cell-cycle, progression and lipid transformation. The Ras-associated binding (Rab) family of GTPases constitutes the largest family of GTPases and consists of almost 70 known members of small GTPases in humans, which are known to play an important role in the regulation of intracellular membrane trafficking, membrane identity, vesicle budding, uncoating, motility and fusion of membranes. Mutations in Rab genes can cause a wide range of inherited genetic diseases, ranging from neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD) to immune dysregulation/deficiency syndromes, like Griscelli Syndrome Type II (GS-II) and hemophagocytic lymphohistiocytosis (HLH), as well as a variety of cancers. Here, we provide an extended overview of human Rabs, discussing their function and diseases related to Rabs and Rab effectors, as well as focusing on effects of (aberrant) Rab expression. We aim to underline their importance in health and the development of genetic and malignant diseases by assessing their role in cellular structure, regulation, function and biology and discuss the possible use of stem cell gene therapy, as well as targeting of Rabs in order to treat malignancies, but also to monitor recurrence of cancer and metastasis through the use of Rabs as biomarkers. Future research should shed further light on the roles of Rabs in the development of multifactorial diseases, such as diabetes and assess Rabs as a possible treatment target.

G-CSF treatment of healthy pediatric donors affects their hematopoietic microenvironment through changes in bone marrow plasma cytokines and stromal cells.

Although G-CSF mobilized peripheral blood stem cell (PBSC) transplantation is commonly used in adults, bone marrow (BM) is still the preferred stem cell source in pediatric stem cell transplantation. Despite the fact that G-CSF is increasingly being used to enhance the hematopoietic stem/progenitor cell (HSPC) yield in BM transplantation (G-BM), the direct effects of G-CSF on the pediatric BM microenvironment have never been investigated. The BM hematopoietic niche provides the physical space where the HSPCs reside. This BM niche regulates HSPC quiescence and proliferation through direct interactions with other niche cells, including Mesenchymal Stromal Cells (MSCs). These cells have been shown to secrete a wide range of hematopoietic cytokines (CKs) and growth factors (GFs) involved in differentiation, retention and homing of hematopoietic cells. Here, we assessed changes in the BM microenvironment by measuring levels of 48 different CKs and GFs in G-BM and control BM (C-BM) plasma from pediatric donors. In addition, the effect of G-CSF on cell numbers and characteristics of HSPCs and MSCs was assessed. IL-16, SCGF-b, MIP-1b (all >1000 pg/mL) and RANTES (>10.000 pg/mL) were highly expressed in healthy donor pediatric BM plasma. Levels of IL-3, IL-18, GROa, MCP-3 (p<0.05) were increased in G-BM, whereas levels of RANTES (p<0.001) decreased after G-CSF treatment. We found a negative correlation with increasing age for IL2-Ra and LIF (p<0.05). In addition, a concomitant increase in the number of both hematopoietic and fibroblast colony forming units was observed, indicating that G-CSF affects both HSPC and MSC numbers. In conclusion, G-CSF treatment of healthy pediatric donors affects the hematopoietic BM microenvironment by expansion of HSPC and MSC numbers and modifying local CK and GF levels.

Stem Cell Applications in Lysosomal Storage Disorders: Progress and Ongoing Challenges.

Lysosomal storage disorders (LSDs) are rare inborn errors of metabolism caused by defects in lysosomal function. These diseases are characterized by accumulation of completely or partially degraded substrates in the lysosomes leading to cellular dysfunction of the affected cells. Currently, enzyme replacement therapies (ERTs), treatments directed at substrate reduction (SRT), and hematopoietic stem cell (HSC) transplantation are the only treatment options for LSDs, and the effects of these treatments depend strongly on the type of LSD and the time of initiation of treatment. However, some of the LSDs still lack a durable and curative treatment. Therefore, a variety of novel treatments for LSD patients has been developed in the past few years. However, despite significant progress, the efficacy of some of these treatments remains limited because these therapies are often initiated after irreversible organ damage has occurred.Here, we provide an overview of the known effects of LSDs on stem cell function, as well as a synopsis of available stem cell-based cell and gene therapies that have been/are being developed for the treatment of LSDs. We discuss the advantages and disadvantages of use of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and induced pluripotent stem cell (iPSC)-related (gene) therapies. An overview of current research data indicates that when stem cell and/or gene therapy applications are used in combination with existing therapies such as ERT, SRT, and chaperone therapies, promising results can be achieved, showing that these treatments may result in alleviation of existing symptoms and/or prevention of progression of the disease. All together, these studies offer some insight in LSD stem cell biology and provide a hopeful perspective for the use of stem cells. Further development and improvement of these stem cell (gene) combination therapies may greatly improve the current treatment options and outcomes of patients with a LSD.

SUL-109 Protects Hematopoietic Stem Cells from Apoptosis Induced by Short-Term Hypothermic Preservation and Maintains Their Engraftment Potential.

The newly developed 6-hydroxychromanol derivate SUL-109 was shown to provide protection during hypothermic storage of several cell lines, but has not been evaluated in hematopoietic stem cells (HSCs). Hypothermic preservation of HSCs would be preferred over short-term cryopreservation to prevent cell loss during freezing/thawing and would be particularly useful for short-term storage, such as during conditioning of patients or transport of HSC transplants. Here we cultured human CD34+ umbilical cord blood (UCB) cells and lineage-depleted (Lin-) Balb/c bone marrow (BM) cells for up to 7 days in serum-free HSC expansion medium with hematopoietic growth factors. SUL-109-containing cultures were stored at 4°C for 3 to 14 days. The UCB cells were tested for viability, cell cycle, and reactive oxygen species (ROS). DMSO-cryopreserved Lin- BM cells or Lin- BM cells maintained for 14 days at 4°C were transplanted into RAG2-/- Balb/c mice and engraftment was followed for 6 months. The addition of SUL-109 during the hypothermic storage of expanded CD34+ UCB cells provided a significant improvement in cell survival of the immature CD34+/CD38- fraction after 7 days of hypothermic storage through scavenging of hypothermia-induced ROS and was able to preserve the multilineage capacity of human CD34+ UCB cells for up to 14 days of cold storage. In addition, SUL-109 protected murine BM Lin- cells from 14 days of hypothermic preservation and maintained their engraftment potential after transplantation in immune-deficient RAG2-/- mice. Our data indicate that SUL-109 is a promising novel chemical for use as a protective agent during cold storage of human and murine HSCs to prevent hypothermia-induced apoptosis and promote cell viability.

Neurological Regulation of the Bone Marrow Niche.

The bone marrow (BM) hematopoietic niche is the microenvironment where in the adult hematopoietic stem and progenitor cells (HSPCs) are maintained and regulated. This regulation is tightly controlled through direct cell-cell interactions with mesenchymal stromal stem (MSCs) and reticular cells, adipocytes, osteoblasts and endothelial cells, through binding to extracellular matrix molecules and through signaling by cytokines and hematopoietic growth factors. These interactions provide a healthy environment and secure the maintenance of the HSPC pool, their proliferation, differentiation and migration. Recent studies have shown that innervation of the BM and interactions with the peripheral sympathetic neural system are important for maintenance of the hematopoietic niche, through direct interactions with HSCPs or via interactions with other cells of the HSPC microenvironment. Signaling through adrenergic receptors (ARs), opioid receptors (ORs), endocannabinoid receptors (CRs) on HSPCs and MSCs has been shown to play an important role in HSPC homeostasis and mobilization. In addition, a wide range of neuropeptides and neurotransmitters, such as Neuropeptide Y (NPY), Substance P (SP) and Tachykinins, as well as neurotrophins and neuropoietic growth factors have been shown to be involved in regulation of the hematopoietic niche. Here, a comprehensive overview is given of their role and interactions with important cells in the hematopoietic niche, including HSPCs and MSCs, and their effect on HSPC maintenance, regulation and mobilization.

Bone marrow mesenchymal stem cell donors with a high body mass index display elevated endoplasmic reticulum stress and are functionally impaired.

Bone marrow mesenchymal stem cells (BM-MSCs) are promising candidates for regenerative medicine purposes. The effect of obesity on the function of BM-MSCs is currently unknown. Here, we assessed how obesity affects the function of BM-MSCs and the role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) therein. BM-MSCs were obtained from healthy donors with a normal (<25) or high (>30) body mass index (BMI). High-BMI BM-MSCs displayed severely impaired osteogenic and diminished adipogenic differentiation, decreased proliferation rates, increased senescence, and elevated expression of ER stress-related genes ATF4 and CHOP. Suppression of ER stress using tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyrate (4-PBA) resulted in partial recovery of osteogenic differentiation capacity, with a significant increase in the expression of ALPL and improvement in the UPR. These data indicate that BMI is important during the selection of BM-MSC donors for regenerative medicine purposes and that application of high-BMI BM-MSCs with TUDCA or 4-PBA may improve stem cell function. However, whether this improvement can be translated into an in vivo clinical advantage remains to be assessed.

B lymphocyte subsets and outcomes in patients with an initial diagnosis of transient hypogammaglobulinemia of infancy.

PURPOSE: Transient hypogammaglobulinemia of infancy (THI) is a common immunodeficiency, but definitive diagnosis can only be made retrospectively. While the pathogenesis is still unknown, abnormalities have been reported in the B cell compartment. In this study, we analysed the B cell subsets of patients with an initial THI diagnosis (n = 20) and compared them with those of healthy age-matched Turkish children (n = 72). METHODS: Flow cytometric analyses of the B subsets were performed by staining with anti-CD27-PE, anti-CD19-PerCP, anti-IgD-FITC and anti-IgM-APC antibodies. RESULTS: During a median follow-up of 6.6 years, 13 patients whose IgG levels had normalized before they reached four years of age were diagnosed with definitive THI. The memory subsets of these patients were lower but not statistically different from the healthy controls (HC). The remaining seven patients had prolonged hypogammaglobulinemia after the age of four and had significantly lower memory B cell subsets compared to the HC. On follow-up, these patients had not experienced recurrent infections or autoimmunity. Re-evaluation of patients' B cell subsets six years later showed that the memory B cell ratios had increased to levels comparable to HC, despite the patients still having mildly low IgG levels. CONCLUSION: Patients with prolonged hypogammaglobulinemia had lower levels of memory B cells despite having a similar clinical course to patients who had been diagnosed with definitive THI. This subgroup of putative THI patients poses a diagnostic and classification dilemma. Our results suggested that these patients' memory B cells and IgG levels may recover over time.

Assessment of non-myelotoxic agents as a preparatory regimen for hematopoietic stem cell gene therapy.

RAG2 deficiency is characterized by a lack of B and T lymphocytes, causing severe lethal infections. Currently, RAG2 deficiency is treated with a Hematopoietic Stem Cell transplantation (HSCT). Most conditioning regimens used before HSCT consist of alkylating myelotoxic agents with or without irradiation and affect growth and development of pediatric patients. Here, we developed a non-myelotoxic regimen using G-CSF, VLA-4I or AMD3100. These agents are known HSC mobilizers or affect bone marrow (BM) permeability and may support the homing of HSCs to the BM, without inducing major side effects. Female Rag2-/- mice were pre-treated with Busulfan (BU), G-CSF, VLA-4I or AMD3100 and transplanted with male BM cells transduced with a lentiviral vector carrying codon optimized human RAG2 (RAG2co). Peripheral blood cell counts increased significantly after G-CSF, VLA-4I and AMD3100 treatment, but not after BU. Reconstitution of PB lymphocytes was comparable for all groups with full immune reconstitution at 6 months post transplantation, despite different methods of conditioning. Survival of mice pre-treated with non-myelotoxic agents was significantly higher than after BU treatment. Here, we show that the non-myelotoxic agents G-CSF, VLA-4I, and AMD3100 are highly effective as conditioning regimen before HSC gene therapy and can be used as an alternative to BU.

Bone marrow mesenchymal stromal cells support regeneration of intestinal damage in a colitis mouse model, independent of their CXCR4 expression.

Inflammatory bowel disease (IBD) is characterized by a chronically dysregulated immune response in the gastrointestinal tract. Bone marrow multipotent mesenchymal stromal cells have an important immunomodulatory function and support regeneration of inflamed tissue by secretion of soluble factors as well as through direct local differentiation. CXCR4 is the receptor for CXCL12 (SDF-1, stromal-derived factor-1) and has been shown to be the main chemokine receptor, required for homing of MSCs. Increased expression of CXCL12 by inflamed intestinal tissue causes constitutive inflammation by attracting lymphocytes but can also be used to direct MSCs to sites of injury/inflammation. Trypsin is typically used to dissociate MSCs into single-cell suspensions but has also been shown to digest surface CXCR4. Here, we assessed the regenerative effects of CXCR4high and CXCR4low MSCs in an immune-deficient mouse model of DSS-induced colitis. We found that transplantation of MSCs resulted in clinical improvement and histological recovery of intestinal epithelium. In contrary to our expectations, the levels of CXCR4 on transplanted MSCs did not affect their regenerative supporting potential, indicating that paracrine effects of MSCs may be largely responsible for their regenerative/protective effects.

Correction of Griscelli Syndrome Type 2 causing mutations in the RAB27A gene with CRISPR/Cas9.

Background/aim: Griscelli Syndrome Type 2 (GS-2) is a rare, inherited immune deficiency caused by a mutation in the RAB27A gene. The current treatment consists of hematopoietic stem cell transplantation, but a lack of suitable donors warrants the development of alternative treatment strategies, including gene therapy. The development of mutation-specific clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing technology has opened the way for custom-designed gene correction of patient-derived stem cells. In this study, we aimed to custom design CRISPR/Cas9 constructs and test their efficiency on homology-directed repair (HDR) on the correction of exon 3 and exon 7 mutations in the RAB27A gene of GS-2 patient-derived mesenchymal stem cells (MSCs) and induced pluripotent stem cells. Materials and methods: We assessed RAB27A gene and protein expression using qRT-PCR, Western Blot, and immune fluorescence in GS-2 patient-derived MSCs and induced pluripotent stem cells (iPSCs). Guide RNAs (gRNAs) and donor DNAs were designed based on patient mutations in exon 3 and exon 7 using the CHOPCHOP online tool and transfected into GS-2 MSCs and iPSCs by electroporation. The cells were cultured for 2 days and then used for mutation analysis using DNA sequencing. Results: MSCs and iPSCs from the GS-2 patients lacked RAB27A gene and protein expression. After gRNA and donor DNAs were designed and optimized, we found HDR efficiency with gRNA3.3 (10% efficiency) and gRNA7.3 (27% efficiency) for MSCs but lower efficiency in iPSCs (<5%). However, transfection of both MSCs and iPSCs resulted in massive cell death, loss of colony formation, and spontaneous differentiation. Conclusion: The use of CRISPR/Cas9 to genetically correct MSCs and iPSCs from GS-2 patients with different mutations through HDR is feasible but requires optimization of the procedure to reduce cell death and improve stem cell function before clinical application.

Gene and Cellular Therapies for Leukodystrophies.

Leukodystrophies are a heterogenous group of inherited, degenerative encephalopathies, that if left untreated, are often lethal at an early age. Although some of the leukodystrophies can be treated with allogeneic hematopoietic stem cell transplantation, not all patients have suitable donors, and new treatment strategies, such as gene therapy, are rapidly being developed. Recent developments in the field of gene therapy for severe combined immune deficiencies, Leber's amaurosis, epidermolysis bullosa, Duchenne's muscular dystrophy and spinal muscular atrophy, have paved the way for the treatment of leukodystrophies, revealing some of the pitfalls, but overall showing promising results. Gene therapy offers the possibility for overexpression of secretable enzymes that can be released and through uptake, allow cross-correction of affected cells. Here, we discuss some of the leukodystrophies that have demonstrated strong potential for gene therapy interventions, such as X-linked adrenoleukodystrophy (X-ALD), and metachromatic leukodystrophy (MLD), which have reached clinical application. We further discuss the advantages and disadvantages of ex vivo lentiviral hematopoietic stem cell gene therapy, an approach for targeting microglia-like cells or rendering cross-correction. In addition, we summarize ongoing developments in the field of in vivo administration of recombinant adeno-associated viral (rAAV) vectors, which can be used for direct targeting of affected cells, and other recently developed molecular technologies that may be applicable to treating leukodystrophies in the future.

Keratinocyte growth factor and stem cell factor to improve thymopoiesis after autologous CD34+ cell transplantation in rhesus macaques.

Deficient thymopoiesis and retarded recovery of naive CD4(+) T cells are important determinants of insufficient immune-competence following hematopoietic stem cell transplantation (HSCT). Although keratinocyte growth factor (KGF) may protect the thymic epithelium, stem cell factor (SCF) is involved in early thymopoiesis. We evaluated whether KGF alone or combined with SCF would affect thymopoiesis and hematologic recovery following myeloablative autologous HSCT into rhesus macaques. Purpose-bred adult rhesus macaques received 10(6) autologous CD34(+)-selected mononuclear bone marrow cells (BMC)/kg after 9 Gy myeloablative conditioning. Animals were treated with phosphate-buffered saline (PBS) (n = 2), KGF alone (n = 2), or KGF combined with SCF (n = 2). KGF-treated animals showed accelerated hematologic recovery, improved thymopoiesis, and enhanced naive T-cell recovery following transplantation. Improved T cell recovery was not associated with protection against cytomegalovirus reactivation nor with improved antibody response to tetanus toxoid vaccination. Animals treated with KGF and SCF experienced severe adverse events that precluded evaluation of thymopoiesis and T cell recovery. Collectively, our data confirm that KGF may enhance thymopoiesis.

Lentiviral gene therapy of murine hematopoietic stem cells ameliorates the Pompe disease phenotype.

Pompe disease (acid alpha-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector-mediated expression of human acid alpha-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid alpha-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid alpha-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue.

Generation and Hematopoietic Differentiation of Mesenchymal Stromal/Stem Cell-Derived Induced Pluripotent Stem Cell Lines for Disease Modeling of Hematopoietic and Immunological Diseases.

Here, we describe a protocol for reprogramming of bone marrow-derived multipotent mesenchymal stromal/stem cells to obtain induced pluripotent stem cells from patients with primary immune deficiencies using lentiviral vectors, followed by hematopoietic differentiation of the MSC-derived iPSCs. This protocol is particularly helpful in cases where it is difficult to obtain sufficient numbers of hematopoietic cells for research and can be applied to model any hematological/immunological disease.

Global miRNA expression of bone marrow mesenchymal stem/stromal cells derived from Fanconi anemia patients.

Fanconi anemia (FA) is a rare genetic disorder characterized by genomic instability, developmental defects, and bone marrow (BM) failure. Hematopoietic stem cells (HSCs) in BM interact with the mesenchymal stem/stromal cells (MSCs); and this partly sustains the tissue homeostasis. MicroRNAs (miRNAs) can play a critical role during these interactions possibly via paracrine mechanisms. This is the first study addressing the miRNA profile of FA BM-MSCs obtained before and after BM transplantation (preBMT and postBMT, respectively). Non-coding RNA expression profiling and quality control analyses were performed in Donors (n = 13), FA preBMT (n = 11), and FA postBMT (n = 6) BM-MSCs using GeneChip miRNA 2.0 Array. Six Donor-FA preBMT pairs were used to identify a differentially expressed miRNA expression signature containing 50 miRNAs, which exhibited a strong correlation with the signature obtained from unpaired samples. Five miRNAs (hsa-miR-146a-5p, hsa-miR-148b-3p, hsa-miR-187-3p, hsa-miR-196b-5p, and hsa-miR-25-3p) significantly downregulated in both the paired and unpaired analyses were used to generate the BM-MSCs' miRNA-BM mononuclear mRNA networks upon integration of a public dataset (GSE16334; studying Donor versus FA samples). Functionally enriched KEGG pathways included cellular senescence, miRNAs, and pathways in cancer. Here, we showed that hsa-miR-146a-5p and hsa-miR-874-3p were rescued upon BMT (n = 3 triplets). The decrease in miR-146a-5p was also validated using RT-qPCR and emerged as a strong candidate as a modulator of BM mRNAs in FA patients.

Strategies to Protect Hematopoietic Stem Cells from Culture-induced Stress Conditions.

In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of hematopoietic stem cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of the available strategies that may be used to protect HSCs from culture-induced stress conditions.

Effects of storage media, supplements and cryopreservation methods on quality of stem cells.

Despite a vast amount of different methods, protocols and cryoprotective agents (CPA), stem cells are often frozen using standard protocols that have been optimized for use with cell lines, rather than with stem cells. Relatively few comparative studies have been performed to assess the effects of cryopreservation methods on these stem cells. Dimethyl sulfoxide (DMSO) has been a key agent for the development of cryobiology and has been used universally for cryopreservation. However, the use of DMSO has been associated with in vitro and in vivo toxicity and has been shown to affect many cellular processes due to changes in DNA methylation and dysregulation of gene expression. Despite studies showing that DMSO may affect cell characteristics, DMSO remains the CPA of choice, both in a research setting and in the clinics. However, numerous alternatives to DMSO have been shown to hold promise for use as a CPA and include albumin, trehalose, sucrose, ethylene glycol, polyethylene glycol and many more. Here, we will discuss the use, advantages and disadvantages of these CPAs for cryopreservation of different types of stem cells, including hematopoietic stem cells, mesenchymal stromal/stem cells and induced pluripotent stem cells.

CXCR4 expression by mesenchymal stromal cells is lost after use of enzymatic dissociation agents, but preserved by use of non-enzymatic methods.

In recent years, multipotent mesenchymal stromal cells (MSCs) have demonstrated tremendous potential for use in regenerative medicine. CXCR4, the receptor for CXCL12, is highly expressed by bone marrow (BM) MSCs and the CXCR4/CXCL12 axis has been shown to be important for migration and homing of BM-MSCs. Typically, MSCs used for clinical applications are collected after culture expansion using enzymatic methods, such as trypsin. Here, we compared different commercially available enzymatic and non-enzymatic methods for collection and dissociation of MSCs from culture plastics and their effects on CXCR4 expression by MSCs. We found that whereas non-enzymatic dissociation buffers and methods maintained CXCR4 expression, all tested enzymatic dissociation solutions dramatically decreased expression of CXCR4. We, therefore, strongly recommend the use of non-enzymatic dissociation methods, followed by filtration through a cell strainer to obtain single cell suspensions, in order to preserve maximal CXCR4 expression and optimal homing of cells.

Development, characterization, and hematopoietic differentiation of Griscelli syndrome type 2 induced pluripotent stem cells.

BACKGROUND: Griscelli syndrome type 2 (GS-2) is a rare, autosomal recessive immune deficiency syndrome caused by a mutation in the RAB27A gene, which results in the absence of a protein involved in vesicle trafficking and consequent loss of function of in particular cytotoxic T and NK cells. Induced pluripotent stem cells (iPSC) express genes associated with pluripotency, have the capacity for infinite expansion, and can differentiate into cells from all three germ layers. They can be induced using integrative or non-integrative systems for transfer of the Oct4, Sox2, Klf4, and cMyc (OSKM) transcription factors. To better understand the pathophysiology of GS-2 and to test novel treatment options, there is a need for an in vitro model of GS-2. METHODS: Here, we generated iPSCs from 3 different GS-2 patients using lentiviral vectors. The iPSCs were characterized using flow cytometry and RT-PCR and tested for the expression of pluripotency markers. In vivo differentiation to cells from all three germlines was tested using a teratoma assay. In vitro differentiation of GS-2 iPSCs into hematopoietic stem and progenitor cells was done using Op9 feeder layers and specified media. RESULTS: All GS-2 iPSC clones displayed a normal karyotype (46XX or 46XY) and were shown to express the same RAB27A gene mutation that was present in the original somatic donor cells. GS-2 iPSCs expressed SSEA1, SSEA4, TRA-1-60, TRA-1-81, and OCT4 proteins, and SOX2, NANOG, and OCT4 expression were confirmed by RT-PCR. Differentiation capacity into cells from all three germ layers was confirmed using the teratoma assay. GS-2 iPSCs showed the capacity to differentiate into cells of the hematopoietic lineage. CONCLUSIONS: Using the lentiviral transfer of OSKM, we were able to generate different iPSC clones from 3 GS-2 patients. These cells can be used in future studies for the development of novel treatment options and to study the pathophysiology of GS-2 disease.

Neuropeptide Y is involved in the regulation of quiescence of hematopoietic stem cells.

Differentiation, self-renewal and quiescence of Hematopoietic stem cells (HSCs) is tightly regulated in order to protect the HSCs from the strain of constant cell division and depletion of the stem cell pool. The neurotransmitter Neuropeptide Y (NPY) is released from sympathetic nerves in the bone marrow and has been shown to indirectly affect HSC function through effects on bone marrow (BM) multipotent Mesenchymal Stromal Cells (MSCs), osteoblasts (OBs) and macrophages. Although the absence of NPY has been shown to be accompanied by severe BM impairment and delayed engraftment of HSCs, the direct effects of NPY on HSCs have never been assessed. Here, we aimed to explore the effect of NPY on the regulation of HSCs. All NPY receptors Y1, Y2, Y4 and Y5 were found to be highly expressed on most HSCs and mature hematopoietic cell subsets. In culture, in particularly expression of the Y1 receptor was shown to decrease in time. Doses of 300 nM NPY suppressed HSC proliferation in cell cultures, as confirmed by an increase of HSCs in G0 phase and an increase in the gene expression levels of FOXO3, DICER1, SMARCA2 and PDK1, which all have been shown to play an important role in the regulation of cell quiescence. These data support the idea that NPY may have a direct effect on the regulation of HSC fate by modulating cell quiescence.