Ana-Violeta Fonseca

New insights into the cell biology of hematopoietic progenitors by studying prominin-1 (CD133).

Prominin-1 (alias CD133) has received considerable interest because of its expression by several stem and progenitor cells originating from various sources, including the neural and hematopoietic systems. As a cell surface marker, prominin-1 is now used for somatic stem cell isolation. Its expression in cancer stem cells has broadened its clinical value, as it might be useful to outline new prospects for more effective cancer therapies by targeting tumor-initiating cells. Cell biological studies of this molecule have demonstrated that it is specifically concentrated in various membrane structures that protrude from the planar areas of the plasmalemma. Prominin-1 binds to the plasma membrane cholesterol and is associated with a particular membrane microdomain in a cholesterol-dependent manner. Although its physiological function is not yet determined, it is becoming clear that this cell surface protein, as a unique marker of both plasma membrane protrusions and membrane microdomains, might reveal new aspects of the cell biology of rare stem and cancer stem cells. The aim of this review is to outline the recent discoveries regarding the dynamic reorganization of the plasma membrane of rare CD133+ hematopoietic progenitor cells during cell migration and division.

Prominin-1 (CD133): from progenitor cells to human diseases

Prominin-1 (CD133) is a pentaspan membrane glycoprotein that binds to plasma membrane cholesterol and concentrates selectively in plasma membrane protrusions. In recent years, this molecule has received considerable interest due to its expression in various progenitors, including those derived from the neural and hematopoietic system, as well as in cancer originating from these systems. Prominin-1 is also the target of mutations leading to retinal degeneration. In the future, prominin-1-positive progenitor cells might become clinically significant, particularly with regard to tissue engineering, and prominin-1 itself might reveal some fundamental cell biological aspects concerning the self-renewal capacity of somatic stem cells. Biomedical research has recently been focusing on the biological characterization of stem and progenitor cells. Understanding, and eventually controlling, the self-renewal capacity of these cells as well as their ability to differentiate into mature cells could lead to the development of new cell-based therapeutic strategies. A considerable research effort is thus being made in order to identify the key players responsible for the proliferation, self-renewal and differentiation of stem and progenitor cells. The current strategy to isolate these rare cell subpopulations is based mainly on monoclonal antibodies directed against specific cell surface markers. Searching for such stem cell surface markers, a

Haematopoietic stem cell differentiation promotes the release of prominin‐1/CD133‐containing membrane vesicles—a role of the endocytic–exocytic pathway

The differentiation of stem cells is a fundamental process in cell biology and understanding its mechanism might open a new avenue for therapeutic strategies. Using an ex vivo co‐culture system consisting of human primary haematopoietic stem and progenitor cells growing on multipotent mesenchymal stromal cells as a feeder cell layer, we describe here the exosome‐mediated release of small membrane vesicles containing the stem and cancer stem cell marker prominin‐1 (CD133) during haematopoietic cell differentiation. Surprisingly, this contrasts with the budding mechanism underlying the release of this cholesterol‐binding protein from plasma membrane protrusions of neural progenitors. Nevertheless, in both progenitor cell types, protein–lipid assemblies might be the essential structural determinant in the release process of prominin‐1. Collectively, these data support the concept that prominin‐1‐containing lipid rafts may host key determinants necessary to maintain stem cell properties and their quantitative reduction or loss may result in cellular differentiation.

Polarization and Migration of Hematopoietic Stem and Progenitor Cells Rely on the RhoA/ROCK I Pathway and an Active Reorganization of the Microtubule Network

Understanding the physiological migration of hematopoietic progenitors is important, not only for basic stem cell research, but also in view of their therapeutic relevance. Here, we investigated the role of the Rho kinase pathway in the morphology and migration of hematopoietic progenitors using an ex vivo co-culture consisting of human primary CD34+ progenitors and mesenchymal stromal cells. The addition of the Rho kinase inhibitor Y-27632 led to the abolishment of the uropod and microvillar-like structures of hematopoietic progenitors, concomitant with a redistribution of proteins found therein (prominin-1 and ezrin). Y-27632-treated cells displayed a deficiency in migration. Time-lapse video microscopy revealed impairment of the rear pole retraction. Interestingly, the knockdown of ROCK I, but not ROCK II, using RNA interference (RNAi) was sufficient to cause the referred morphological and migrational changes. Unexpectedly, the addition of nocodazole to either Y-27632- or ROCK I RNAi-treated cells could restore their polarized morphology and migration suggesting an active role for the microtubule network in tail retraction. Finally, we could demonstrate using RNAi that RhoA, the upstream regulator of ROCK, is involved in these processes. Collectively, our data provide new insights regarding the role of RhoA/ROCK I and the microtubules in the migration of stem cells.

Differential expression of biofunctional GM1 and GM3 gangliosides within the plastic-adherent multipotent mesenchymal stromal cell population.

BACKGROUND AIMS It is unclear whether the plastic-adherent multipotent mesenchymal stromal cells (MSC) isolated from human bone marrow (BM) represent a uniform cell population or are heterogeneous in terms of cell-surface constituents and hence functionality. METHODS We investigated the expression profile of certain biofunctional lipids by plastic-adherent MSC, focusing particularly on two membrane microdomain (lipid raft)-associated monosialogangliosides, GM1 and GM3, using indirect confocal laser scanning fluorescence microscopy and flow cytometry. RESULTS Phenotypically, we observed a differential expression where certain MSC subsets exhibited GM1, GM3 or both at the plasma membrane. Furthermore, disialoganglioside GD2 detection increased the complexity of the expression patterns, giving rise to seven identifiable cell phenotypes. Variation of standard culture conditions, such as the number of cell passage and period in culture, as well as donors, did not influence the heterologous ganglioside expression profile. In contrast, the binding of various lectins appeared homogeneous throughout the MSC population, indicating that the general glycosylation pattern remained common. Morphologically, the expression of a given ganglioside-based phenotype was not related to a cell with particular size or shape. Interestingly, a segregation of GM1 and GM3 clusters was observed, GM3 being mostly excluded from the highly curved plasma membrane protrusions. CONCLUSIONS These data highlight the phenotypic heterogeneity of plastic-adherent MSC in terms of certain lipid constituents of the plasma membrane, and the presence and/or absence of distinct ganglioside-based membrane microdomains suggest their potential functional diversity.

The hematopoietic stem cell polarization and migration: A dynamic link between RhoA signaling pathway, microtubule network and ganglioside-based membrane microdomains.

The polarization and migration of eukaryotic cells are fundamental processes for the development and maintenance of a tissue. These aspects gain especial interest when it comes to stem and progenitor cells in the way that their manipulation might open new avenues in regenerative therapy. In recent years, novel biological facets of migrating hematopoietic stem cells were revealed by several groups including ours. Among these features, the polarization of their membranous (proteins and lipids) and cytoplasmic constituents, which leads to the formation of a specialized sub-cellular structure located at the rear pole – the uropod – has gained increasing interest. In a new study we have demonstrated that such phenomena involve a coordinated mechanism between Rho GTPase signaling and the microtubule network. Specifically, our results based on the use of synthetic inhibitors and RNA interference suggest that the activity of RhoA and its effector ROCK I is indispensable for cell polarization and the active reorganization of microtubules that are required for migration.

Migration of Stem Cells: Role of the RhoA/ROCK I Pathway (Method)

Cell migration is an important biological phenomenon that has come under the spotlight following the worldwide emergence of stem cell-based therapies. How a given stem cell migrates within an organism to reach its final destination, known as the stem cell niche, to replenish a cellular system is a question of interest. The development of new cell-isolation and transfection techniques together with ex vivo culture systems have allowed the successful isolation, manipulation and expansion of stem cells. When combined with real-time imaging techniques, novel biochemical tools such as RNA interference oligonucleotides and animal models, have shed new light on the mechanisms that regulate stem cell migration. Here, we summarize the migration mechanisms that are based on biochemical pathways related to Rho GTPases. In particular, we focus on the RhoA/ROCK I pathway that affects the polarization of hematopoietic stem and progenitor cells and therefore the driving forces underlying migration.