A side order of stem cells: The SP phenotype

A defining property of murine hematopoietic stein cells (HSCs) is low fluorescence after staining with Hoechst 33342 and Rhodamine 123. These dyes have proven to be remarkably powerful tools in the purification and characterization of HSCs when used alone or in combination with antibodies directed against stem cell epitopes. Hoechst low cells are described as side population (SP) cells by virtue of their typical profiles in Hoechst red versus Hoechst blue bivariate fluorescent-activated cell sorting dot plots. Recently, excitement has been generated by the findings that putative stem cells from solid tissues may also possess this SP phenotype. SP cells have now been isolated from a wide variety of mammalian tissues based on this same dye efflux phenomenon, and in many cases this cell population has been shown to contain apparently multipotent stem cells. What is yet to be clearly addressed is whether cell fusion accounts for this perceived SP multipotency. Indeed, if low fluorescence after Hoechst staining is a phenotype shared by hematopoietic and organ-specific stem cells, do all resident tissue SP cells have bone marrow origins or might the SP phenotype be a property common to all stem cells? Subject to further analysis, the SP phenotype may prove invaluable for the initial isolation of resident tissue stem cells in the absence of definitive cell-surface markers and may have broad-ranging applications in stem cell biology, from the purification of novel stem cell populations to the development of autologous stem cell therapies.

Brain tumour stem cells

The dogma that the genesis of new cells is a negligible event in the adult mammalian brain has long influenced our perception and understanding of the origin and development of CNS tumours. The discovery that new neurons and glia are produced throughout life from neural stem cells provides new possibilities for the candidate cells of origin of CNS neoplasias. The emerging hypothesis is that alterations in the cellular and genetic mechanisms that control adult neurogenesis might contribute to brain tumorigenesis, thereby allowing the identification of new therapeutic strategies.

Transplantation of mesenchymal stem cells promotes an alternative pathway of macrophage activation and functional recovery after spinal cord injury

Abstract Mesenchymal stem cells (MSC) derived from bone marrow can potentially reduce the acute inflammatory response in spinal cord injury (SCI) and thus promote functional recovery. However, the precise mechanisms through which transplanted MSC attenuate inflammation after SCI are still unclear. The present study was designed to investigate the effects of MSC transplantation with a special focus on their effect on macrophage activation after SCI. Rats were subjected to T9-T10 SCI by contusion, then treated 3 days later with transplantation of 1.0×10(6) PKH26-labeled MSC into the contusion epicenter. The transplanted MSC migrated within the injured spinal cord without differentiating into glial or neuronal elements. MSC transplantation was associated with marked changes in the SCI environment, with significant increases in IL-4 and IL-13 levels, and reductions in TNF-a and IL-6 levels. This was associated simultaneously with increased numbers of alternatively activated macrophages (M2 phenotype: arginase-1- or CD206-positive), and decreased numbers of classically activated macrophages (M1 phenotype: iNOS- or CD16/32-positive). These changes were associated with functional locomotion recovery in the MSC-transplanted group, which correlated with preserved axons, less scar tissue formation, and increased myelin sparing. Our results suggested that acute transplantation of MSC after SCI modified the inflammatory environment by shifting the macrophage phenotype from M1 to M2, and that this may reduce the effects of the inhibitory scar tissue in the subacute/chronic phase after injury to provide a permissive environment for axonal extension and functional recovery.

Microspheres as a vehicle for biomolecule delivery to neural stem cells

Neural stem cells (NSC) are a valuable model system for understanding the intrinsic and extrinsic controls for self-renewal and differentiation choice. They also offer a platform for drug screening and neurotoxicity studies, and hold promise for cell replacement therapies for the treatment of neurodegenerative diseases. Fully exploiting the potential of this experimental tool often requires the manipulation of intrinsic cues of interest using transfection methods, to which NSC are relatively resistant. In this paper, we show that mouse and human NSC readily take up polystyrene-based microspheres which can be loaded with a range of chemical or biological cargoes. This uptake can take place in the undifferentiated stage without affecting NSC proliferation and their capacity to give rise to neurons and glia. We demonstrate that ß-galactosidase-loaded microspheres could be efficiently introduced into NSC with no apparent toxic effect, thus providing proof-of-concept for the use of microspheres as an alternative biomolecule delivery system.

Changes in gene expression and morphology of mouse embryonic stem cells on differentiation into insulin-producing cells in vitro and in vivo

Background Embryonic stem (ES) cells have the potential to produce unlimited numbers of surrogate insulin-producing cells for cell replacement therapy of type I diabetes mellitus. The impact of the in vivo environment on mouse ES cell differentiation towards insulin-producing cells was analysed morphologically after implantation. Methods ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new four-stage differentiation protocol were analysed morphologically before and after implantation for gene expression by in situ reverse transcription polymerase chain reaction and protein expression by immunohistochemistry and ultrastructural analysis. Results In comparison with nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the four-stage protocol increased under in vivo conditions upon morphological analysis. The cells exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, islet amyloid polypeptide (IAPP), the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a four-stage protocol enabled further significant maturation for the beta-cell-specific markers, insulin and the co-stored IAPP as well as the glucose recognition structures. in contrast, further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol. Conclusions A sufficient degree of in vitro differentiation is an essential prerequisite for further substantial maturation in a beta-cell-specific way in vivo, supported by cell-cell contacts and vascularisation. Copyright (c) 2009 John Wiley & Sons, Ltd.

A new experimental protocol for preferential differentiation of mouse embryonic stem cells into insulin-producing cells

Mouse embiyonic stem (ES) cells have the potential to differentiate into insulin-producing cells, but efficient protocols for in vitro differentiation have not been established. Here we have developed a new optimized four-stage differentiation protocol and compared this with an established reference protocol. The new protocol minimized differentiation towards neuronal progeny, resulting in a population of insulin-producing cells with ß-cell characteristics but lacking neuronal features. The yield of glucagon and somatostatin cells was negligible. Crucial for this improved yield was the removal of a nestin selection step as well as removal of culture supplements that promote differentiation towards the neuronal lineage. Supplementation of the differentiation medium with insulin and fetal calf serum was beneficial for differentiation towards monohor-monal insulin-positive cells. After implantation into diabetic mice these insulin-producing cells produced a time-dependent improvement of the diabetic metabolic state, in contrast to cells differentiated according to the reference protocol. Using a spinner culture instead of an adherent culture of ES cells prevented the differentiation towards insulin-producing cells. Thus, prevention of cell attachment in a spinner culture represents a means to keep ES cells in an undifferentiated state and to inhibit differentiation. In conclusion, this study describes a new optimized four-stage protocol for differentiating ES cells to insulin-producing cells with minimal neuronal cell formation. Copyright © 2008 Cognizant Comm. Corp.

Bone marrow-derived mesenchymal stem cells become anti-angiogenic when chondrogenically or osteogenically differentiated:implications for bone and cartilage tissue engineering

Osteochondral tissue repair requires formation of vascularized bone and avascular cartilage. Mesenchymal stem cells stimulate angiogenesis both in vitro and in vivo but it is not known if these proangiogenic properties change as a result of chondrogenic or osteogenic differentiation. We investigated the angiogenic/antiangiogenic properties of equine bone marrow-derived mesenchymal stem cells (eBMSCs) before and after differentiation in vitro. Conditioned media from chondrogenic and osteogenic cell pellets and undifferentiated cells was applied to endothelial tube formation assays using Matrigel™. Additionally, the cell secretome was analysed using LC-MS/MS mass spectrometry and screened for angiogenesis and neurogenesis-related factors using protein arrays. Endothelial tube-like formation was supported by conditioned media from undifferentiated eBMSCs. Conversely, chondrogenic and osteogenic conditioned media was antiangiogenic as shown by significantly decreased length of endothelial tube-like structures and degree of branching compared to controls. Undifferentiated cells produced higher levels of angiogenesis-related proteins compared to chondrogenic and osteogenic pellets. In summary, eBMSCs produce an array of angiogenesis-related proteins and support angiogenesis in vitro via a paracrine mechanism. However, when these cells are differentiated chondrogenically or osteogenically, they produce a soluble factor(s) that inhibits angiogenesis. With respect to osteochondral tissue engineering, this may be beneficial for avascular articular cartilage formation but unfavourable for bone formation where a vascularized tissue is desired. © Copyright 2014, Mary Ann Liebert, Inc.