Eliana Perissinotto

Fast But Durable Megakaryocyte Repopulation and Platelet Production in NOD/SCID Mice Transplanted with Ex-Vivo Expanded Human Cord Blood CD34+ Cells

We have previously established a stroma‐free culture with Flt‐3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) that allows the maintenance and the expansion for several weeks of a cord blood (CB) CD34+ cell population capable of multilineage and long‐lasting hematopoietic repopulation in non‐obese diabetic/ severe combined immunodeficient (NOD/SCID) mice.

Detection ofBCL-6 rearrangements andp53 mutations in malt-lymphomas

Twenty‐seven lymphomas of mucosa‐associated lymphoid tissue (MALT) derived from distinct anatomical sites were tested for the presence of genetic lesions commonly involved in B‐cell lymphomagenesis, including activation of proto‐oncogenes (BCL‐1, BCL‐2, BCL‐6, and c‐MYC), disruption of tumor suppressor loci (p53, 6q), and infection by viruses [Epstein‐Barr virus (EBV), and Kaposis sarcoma‐herpesvirus/human herpesvirus‐8 (KSHV/HHV‐8)]. Sixteen low‐grade and 11 high‐grade MALT‐lymphomas were included in the study. The presence of genetic lesions was tested by a combination of molecular approaches, including Southern blot hybridization, polymerase chain reaction (PCR), and PCR‐single strand conformation polymorphism followed by DNA direct sequencing. Alterations of BCL‐1, BCL‐2, or c‐MYC, as well as infection by KSHV/HHV‐8, scored negative in all MALT‐lymphomas analysed. Conversely, rearrangements of BCL‐6 and mutations of p53 clustered with a fraction of high‐grade MALT‐lymphomas. Deletions of 6q occurred in selected cases of both low‐ and high‐grade MALT‐lymphomas, whereas a monoclonal infection by EBV was restricted to one single patient. These data corroborate the notion that the molecular pathogenesis of MALT‐lymphomas differs substantially from that of nodal B‐cell lymphomas. Occasionally, however, a proportion of high‐grade MALT‐lymphomas may harbor selected genetic lesions among the ones commonly involved in nodal B‐cell lymphomagenesis. Am. J. Hematol. 56:206–213, 1997.

Megakaryocyte differentiation and reconstitution in nod/scid mice of CD34+ cord blood cells after extensive ex vivo expansion

Abstract Safe use of myeloablative regimes is dependent on the availability of grafts containing sufficient number of haemopoietic stem cells to guarantee a durable reconstitution of the entire hematopoietic system. Much interest has been paid to alternative sources of hematopoietic stem cells that could be expanded ex vivo prior to being transplanted. In our hands, cord blood (CB) CD34 + cells can be triggered to undergo extensive proliferation and self-renewal processes, without losing, but, rather, amplifying their in vivo repopulating capacity. A central issue remains whether CD34 + cells, after several weeks of expansion, still retain not only their self-renewal characteristics, but also the same proliferation and differentiation potential towards all of the haemopoietic lineages, among all the megakaryocytic. We compared the proliferation and differentiation capacity toward the Mk lineage, as well as the Mk reconstitution in NOD/SCID mice of fresh, unmanipulated CD34 + cells derived from several cord blood samples and those recovered after extensive expansion in stroma-free cultures supported by FL, TPO and SCF. Unmanipulated CD34 + CB cells were grown in suspension cultures + either 10% FCS or 10% human plasma (HP) with: 1) TPO; 20 TPO + SCF; 3) IL3 + SCF + IL6; 4) IL3 + SCF + IL6 + TPO for 7, 14, 21 and 28 days. Cell production, appearance of CD41 + , CD34 + , CD34 + /CD41 + cells, CFU-Mk output as well as Mk repopulation in NOD/SCID mice were monitored and the indicated time points. The same studies were performed on CD34 + and CD34 − cells obtained by separation procedures (MiniMACS columns or FACS sorting) from CB cells which had been expanded for at least four weeks in stroma-free cultures in the presence of FL, TPO and SCF. In either case cells were cultured in IMDM+ 10% FCS or HP with the 4 described growth factor combinations. TPO by itself triggered maximum Mk production (/up to 500 -fold) in fresh but not in expanded cells, for whom the additional presence of SCF and IL6 was important to generate not only mature Mk, but also more primitive unipotent Mk or multipotent CFU-GEMM progenitors. NOD/SCID mice, studies indicate a normal Mk engraftment of expanded cells. The role of accessory CD34 − cells being investigated).

Role of Hematopoietic Growth Factors on the ex Vivo Expansion of Primitive Cord Blood Stem Cells

Ex vivo expansion of hematopoietic cells has recently been suggested for clinical application.1.2 It has been demonstrated that hematopoietic compartments derived from human bone marrow (BM), mobilized peripheral blood (PB) or umbilical cord blood (CB) cells can be maintained and expanded in liquid culture or stroma coculture systems by the provision of combinations of growth factors.3–5 The potential benefits of such studies include accelerated engraftment, reduced risk of infection, smaller stem cell harvests, and improved effectiveness of genetically modified stem cells.1.2 Although controversies remain concerning what defined populations may or may not be useful for improving in vivo hematologic recovery, ex vivo expansion can only be considered successful when progeny receptor cells retain both pluripotent differentiation and self-renewal capacities of the original stem cells.4 In other words, are the various growth factor combinations able to amplify the late progenitor reservoir without exhausting the stem cell pool, in order to ensure long-term post-transplantation engraftment after a myeloablative conditioning regimen? Human culture systems employing Interleukin 3 (IL3) or Granulocyte-Colony Stimulating Factor (G-CSF) maintain at least some primitive cells;6–9 however, achieving a net expansion of early cells has proven elusive, although some laboratories have reported encouraging results.10–12 With the discovery of new growth factors, specific for early progenitor cells, investigators have re-examined the possibility of expanding stem cells in vitro. In particular, three cytokines discovered in the early 1990’s, c-kit ligand or Stem Cell Factor (KL), flt3 ligand (FL), and c-mpl ligand also termed Megakaryocyte Growth and Development Factor (MGDF) or Thrombopoietin (TPO), appear to have unique activities on primitive progenitor/stem cells.