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.

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.

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.