Device and method to sort totipotent stem cells

Research and use of human embryonic stem cells is either prohibited in many countries or strongly restricted by law or bioethical issues. A limited number of totipotent stem cells is indeed available from other sources. The development of techniques for sorting totipotent stem cells from such sources is, then, increasingly sought. Generally, it is still an open issue the possibility to sort/select totipotent/multipotent, adherent, living, and particularly human stem cells by means of a method that is relatively simple, inexpensive, which does not effect cell viability and differentiation ability, and which can be applied by bench personnel of standard specialization. Mesenchimal stem cells, hereafter referred to as MSCs, are multipotent, adherent stem cells. MSCs are present and isolated from different sources, among which bone marrow, amniotic membrane, chorial membrane, Wharton gel, cord blood and placenta, dental pulp, and lipoaspirates. Although no phenotype differences are observed in the human MSCs obtained from different sources, some preliminary work based on Transmission Electron Microscopy (hereafter referred to as TEM) characterization of MSCs from different human sources have shown that ultrastructural differences of phenotypically similar MSCs derived from different human tissues can be related to their in vitro differentiation potential. However MSCs express a panel of surface antigens that is so rich and diversified to limit the possibility to precisely distinguish and define this population just on a immuno-phenotypical basis. Here the topic of our patent pending is presented. The invention consists in a new device and methods for a tag-less sorting/enrichment of human, living totipotent stem cells from different sources. The method is able to sort cells based on their biophysical and morphological properties. The invented method realizes a flow-gravity-assisted fractionation of the cell sample. Since the flow-assisted fractionation process requires cells being in suspension, the invented method in fact realizes cell fractionation based on the cell biophysical differences acquired in dynamic conditions. The differences in cell features that are dynamically acquired during fractionation allow for cell fractionation. Because of the extremely gentle fractionation conditions, native cell features are fully restored when cells return to the adherent state after fractionation completion, and cell viability is fully preserved. The method will be hereafter referred to as Non-Equilibrium, Earth Gravity-Assisted Dynamic Fractionation (NEEGA-DF). The NEEGA-DF method is suited to employ any transport fluid for cell fractionation. In particular, the cell culture medium can be employed. This ensures optimal treatment of the cells during the fractionation process, with full maintenance of cell viability and highest cell recovery. MSCs cells isolated from different sources and characterized as described above are fractionated with NEEGA-DF method. After fractionation the cells are collected and cultured on proper surfaces to favour the re-adhesion process that is necessary to obtain their in vitro expansion before any further usage. The fractionated cells can either be immediately employed or stored for further usage

Hepatocyte growth factor induces proliferation and differentiation of multipotent hematopoietic progenitors

Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU-GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34+ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment

Mutations of the Fanconi anemia group A (FAA) gene in Italian patients

SummaryFanconi anemia (FA) is an autosomal recessive disease characterized by progressive pancytopenia, congenital malformations, and predisposition to acute myeloid leukemia. At least five complementation groups (FA-A–FA-E) have been identified. The relative prevalence of FA-A has been estimated at an average of ∼65% but may widely vary according to ethnic background. In Italy, 11 of 12 patients analyzed by cell-fusion studies were assigned to group FA-A, suggesting an unusually high relative prevalence of this FA subtype in patients of Italian ancestry. We have screened the 43 exons of the FAA gene and their flanking intronic sequences in 38 Italian FA patients, using RNA-SSCP. Ten different mutations were detected: three nonsense and one missense substitutions, four putative splice mutations, an insertion, and a duplication. Most of the mutations are expected to cause a premature termination of the FAA protein at various sites throughout the molecule. Four protein variants were also found, three of which were polymorphisms. The missense mutation D1359Y, not found in chromosomes from healthy unrelated individuals, was responsible for a local alteration of hydrophobicity in the FAA protein, and it was likely to be pathogenic. Thus, the mutations so far encountered in the FAA gene are essentially all different. Since screening based on the analysis of single exons by genomic DNA amplification apparently detects only a minority of the mutations, methods designed to detect alterations in the genomic structure of the gene or in the FAA polypeptide may be helpful in the identification of FAA mutations

Clinical implications of the heterogeneity of hematopoietic progenitors elicited in peripheral blood by anticancer therapy with cyclophosphamide and cytokine(s)

Clinical investigators have found that the hematopoietic system irreversibly damaged by cancer therapy with myeloablative high doses of chemoradiotherapy can be reconstituted by transplantation of autologous hematopoietic progenitors retrieved from peripheral blood. In comparison with patients transplanted with bone marrow, those who receive peripheral blood progenitors undergo shorter periods of neutropenia and thrombocytopenia, require less platelet and erythrocyte transfusions and, most importantly, experience overall reduced treatment-related morbidity. In this article, we speculate that an explanation for this clinical achievement may be that committed hematopoietic progenitors as well as ancestral uncommitted pluripotent stem cells are retrieved from circulation and transplanted after myeloablative cancer therapy. As indicated by studies in rodents, transplantation of hematopoietic progenitors is followed by two phases of engraftment associated with progenitors at different stages of maturation. An initial phase corresponding to early hematopoietic recovery is produced by committed progenitors, and a second sustained engraftment phase is produced by the pluripotent stem cell. Should this multiphase engraftment model be true of humans also, the exceptionally prompt and sustained blood cell count recovery achieved by transplanting blood progenitor cells may reflect transplantation of heterogeneous progenitors such as committed progenitors and pluripotent stem cells producing an early engraftment phase and then sustained hematopoiesis, respectively

In vivo and in vitro inhibitory effect of alpha-interferon on megakaryocyte colony growth in essential thrombocythaemia.

Megakaryocyte (MK) colony growth of bone marrow mononuclear non-adherent cells was evaluated in 28 patients with essential thrombocythaemia (ET) and in 26 normal controls. The number of MK-colony forming units (CFU-MK per 3 x 10(5) plated cells) was similar in ET (68 +/- 33) and in controls (63 +/- 37), independently of bone marrow accessory cells. On the contrary, the size of the MK colonies was significantly (P less than 0.01) greater in ET patients. Human recombinant alpha-interferon 2a (alpha-IFN), administered to 10 patients at a dose of 3 x 10(6) IU/d s.c. for 11 +/- 3 weeks, was capable of inducing a significant (P less than 0.01) decrease in the number (from 72 +/- 16 to 31 +/- 14) and size of bone marrow CFU-MK, together with a significant reduction of the platelet count (from 1031 +/- 325 to 378 +/- 75 x 10(9)/l). When added in vitro at time 0 to the culture dishes, alpha-IFN inhibited the CFU-MK growth of both normal and ET bone marrow samples, even at very low concentrations (1 and 10 IU/ml). This study demonstrates that alpha-IFN, both in vivo and in vitro, exerts an inhibitory effect on the growth of MK progenitors, which appears to correlate with the clinically documented antiproliferative effect of this cytokine