Sandro Eridani

Erythropoietic colonies in a serum‐free system: results in primary proliferative polycythaemia and thrombocythaemia

Summary. The formation of erythropoietic colonies from the peripheral blood of normal subjects, patients with primary proliferative polycythaemia (PPP) and primary thrombocythaemia (PT) was studied, using a chemically defined serum‐free (S ‐) medium. Colony formation was markedly more prominent in the presence of burst‐promoting activity (BPA) and erythropoietin (Ep) than with medium alone (P<0.001). In cultures using medium alone, significantly more PPP patients formed colonies than the control group (P<0.05). In the PT group this difference did not achieve statistical significance, but the mean BFU‐E yield was significantly greater than in controls (P<0.05). In a separate series of experiments, parallel cultures in serum‐containing (S +) and serum‐free (S ‐) systems, in the presence of BPA and Ep did not show any significant difference in colony yield. The growth of‘endogenous’colonies in cultures with serum‐free medium alone could be due to a peculiar sensitivity of erythropoietic progenitors to growth factors other than Ep.

Erythroid colony formation in primary thrombocythaemia: evidence of hypersensitivity to erythropoietin

Summary In a group of 14 patients with primary thrombocythaemia (PT) the study of erythropoietic colony formation in vitro showed the development of so‐called ‘endogenous’colonies, namely colonies with no added erythropoietin (Ep), in all but one case. In the presence of added Ep the colony formation increased slightly in the PT group, but the rise in the control group was so pronounced that any statistical difference between the two groups disappeared. No ‘endogenous’colonies were observed in cases of secondary thrombocytosis. This finding reinforces the view that PT is a myeloproliferative disorder and establishes that some erythroid progenitors show the same high sensitivity to Ep as that found in polycythaemia vera; it is of interest that, in the patients studied, there was no apparent involvement of erythropoiesis.

Cytokine effect on ex vivo expansion of haemopoietic stem cells from different human sources

Human pluripotential stem cells (PSC) are currently the target for transplantation attempts and genetic manipulation. We have therefore investigated the frequency and the expansion potential of PSC’s in different types of blood samples. CD 34+ cells were thus obtained from human bone marrow (BM), as well as from peripheral blood (PB) and cord blood (CB) samples. After immuno-magnetic separation the highest yields of CD 34+ cells were from BM (1.08–2.25%) and CB (0.42–1.32%) while PB samples gave much lower values. Suspension cultures of PSC’s from the three sources were then set up, in the presence of combinations of haemopoietic growth factors. A remarkable amplification of the nucleated cell pool was observed reaching a maximum between 10 and 15 days of culture; earliest and maximum expansion (up to 220-fold) was achieved when Erythropoietin (Epo) was added to the culture medium, but this resulted in reduction of colony-forming cells and differentiation into erythroid progenitors. Clonogenic tests for BFU-E’s derived colonies showed a peak value at 5 days of liquid culture. Further studies are advisable to establish the best cytokine combination for a valuableex vivo expansion, coupled with preservation of stem cell properties.

Fetal hemoglobin reactivation and cell engineering in the treatment of sickle cell anemia

The natural history of severe hemoglobinopathies like sickle cell disease (SCD) is rather variable, depending on the circumstances, but the main influence on such variability is the level of fetal hemoglobin (HbF) in the patient’s red cells. It is well known that a significant HbF level is associated with a milder course of disease and fewer complications. Therefore, attempts have been made to reactivate using various means the HbF production, which is normally switched off perinatally. A pharmacological approach has been attempted since the 1980s, ranging from drugs like 5-azacytidine and its derivative, decitabine, to a series of compounds like hydroxyurea and a number of histone deacetylase inhibitors like butyrate, which seem to act as epigenetic modifiers. Many other disparate agents have been tried with mixed results, but hydroxyurea remains the most effective compound so far available. Combinations of different compounds have also been tried with some success. Established treatments like bone marrow or cord blood transplantation are so far the only real cure for a limited number of patients with severe hemoglobinopathies. Improved chemotherapy regimens of milder toxicity than those employed in the past have made it possible recently to obtain a stable, mixed donor-recipient chimerism, with reversal of the SCD phenotype. However, great effort is directed to cell engineering, searching for an effective gene vector by which a desired gene can be transferred into new classes of vectors for autologous hemopoietic stem cells. Recent studies are also aiming at targeted insertion of the therapeutic gene into hemopoietic cells, which can also be “induced” human stem cells, obtained from somatic dedifferentiated cells. Attention in this area must be paid to the possibility of undesired effects, like the emergence of potentially oncogenic cell populations. Finally, an update is presented on improved HbF determination methods, because common international standards are becoming mandatory.

Stem cells: From embryology to cellular therapy? An appraisal of the present state of art

AbtractA series of publications has dealt in the last years with topics as the isolation, properties and applications of animal stem cells (Weissman 2000. Cell 100: 157–168; Weissman 2002. N. Engl. J. Med. 346: 1567–1579; Lovell-Badge 2001. Nature 414: 88–91; Marshak et al. 2001. Stem Cell Biology. Cold Spring Harbor Laboratory Press, New york; Eridani 2002. J. Roy. Soc. Med. 95: 5–8; Borge and Evers 2003. Cytotechnology 41: 59–68; Sgaramella 2003. Cytotechnology 41: 69–73), however, the bonanza of experimental data recently accumulating have raised such an amount of controversial views and discussions that time perhaps has come for a reassessment of the basic facts in this peculiar area of research and an evaluation of possible, not unrealistic, implications.

Mammalian artificial chromosomes: A review.

A mammalian artificial chromosome (MAC) may be assembled through the juxtapposition of three kinds of DNA elements: a centromere, several DNA replication origins, and two telomeric repeats. The resulting structure should be able to carry and express one or more selected genes (transgenes), introduced for specific purposes. The minimal length is unknown, but may be of several Mb.Of its basic elements, the telomeres may present lesser problems, in view of their simple composition and organization. Centromeres could be an issue, given their many unknowns. Mammalian DNA replication origins are at present poorly characterized, but it is expected that at least one may be contained within the MAC components, especially the transgene. Their overall assembly may require a combination of in vivo and in vitro approaches.A promising strategy aims at constructing two telomeric arms of a MAC, one of which may include the transgene. The two novel arms could acquire a functional centromere through recombination with the two arms of a resident chromosome. Alternatively, if the two telomeric constructs are also endowed with properly placed and oriented centromeric sequences, a centromere may be rescued in vivo by homologous recombination with the external parts of the centromere of the resident chromosome. Positive selection for the artificial arms and counterselection against the resident arms should facilitate the assembly process.The assembly of such construct would not change the ploidy number of the host cell. After loading of a transgene, however, the resulting MAC may be isolated and transferred into an expression cell, where it may represent a novel chromosomal element. In this case untoward effects to the host cell may derive from an ensuing dosage effect for the transgene(s) rather than from the presence of a MAC per se.A MAC may contribute to a deeper understanding of the structural requirements for chromosomal function and evolution as well as the mechanism of chromatin formation. It should also help in the development of second generation vectors for transfer of Mb-long DNA sequences, as required for properly regulated mammalian gene function as well as, possibly, for therapy.

Stem cell research: State of the art

The present issue aims to provide a general evaluationof the state of the art in a field, which is surroundedby a very unusual attention by all quarters of the sci-entific, social and political arena. The reason why allthis is happeningis of course related to the outstandingdevelopments in stem cell research, possibly leadingto applications in human biology and medicine, whichwere unthinkable only a few years ago.We want therefore to present an updated and ob-jective series of reviews of the state of the art in thisarea: is however important to point out that, in aseries of papers on related subjects, a modest degreeof overlapping topics and considerations is inevitable.We also have not avoided controversial issues, but ofcourseanyopinionis theresponsabiltyof thescientistsactually involved. We hope that this work will helpinterested people to form their own judgment on suchproblems.A very early definition of stem cells was of ‘an-cestral cells of the germ line’ (Wilson 1898). Later,however, it became apparent that stem cells are notconfined to the germ line and that they are endowedwith specific properties; therefore Laitha proposed in1970 a classical definition: ‘cells which have the ca-pacity to divide simmetrically to expand their numberand asimmetrically in order to self-renew and give riseto a differentiated progeny’.A variety ofrecent reviewshavedealt with the gen-eral topic of stem cell characterization, differentiationand versatility (Van der Kooy and Weiss, 2000; Snyderand Vescovi, 2000; Weissman, 2000; Eridani, 2001;Blau et al., 2001; Lovell-Badge, 2001).An updated review of mathematical and experi-mental models for stem cell renewal and differenti-ation is presented in this issue by Viswanathan andZandstra.A significant, very recent advance has been therecognition that both murine and human stem cellsexpress a higher number of genes than differentiatedcells, although their function is often still unknown(Ramalho Santos et al., 2002). Moreover, they share avarietyofexpressedgeneproducts, definingconservedregulatory pathways; in mice the genetic programmeof haemopoietic stem cells is shared with embryonicand neural stem cells. It is suggested that this set ofgeneproductsmayrepresenta ‘signature’ofstem cells(Ivanova et al., 2002).Among the germ cells, blastocyst cells do not self-renew and are not present over the entire lifespan ofthe body; also, oocytes are produced in a limited num-ber shortly after birth. Therefore, according to Vander Kooy and Weiss (2000), such elements cannot becalled stem cells: only spermatogonial cells satisfy therequirement for real stem cells.Embryonic Stem Cells are multipotent stem cellswhich can be derived from the blastocyst during theearly stages of mammalian development. There aretwo types of embryonic cells: EG (Embryonic Germ),obtained from primordial germ cells taken from thedeveloping gonadal ridges of human fetuses and ES,derived from the inner cell mass of preimplantationembryos. ES can be indefinitely maintained in an un-differentiated state, can colonize all tissues includinggermline and they can originate, using transgenic andgene targeting technology, animal models for diseases(Smith, 2001).Human ES express also a wide range of differ-entiation markers, can proliferate for long time inculture, can be cryopreserved and clonally isolated.They can thus be used to derive a great variety of celltypes, including cells of endodermal, mesodermal andectodermal origin (Shamblott et al., 2001).The characteristics and the possible use of em-bryonic stem cells are extensively discussed in this

Erythropoietin gene expression in haemopoietic cell lines.

Erythropoietin (Epo) gene expression was studied in a number of different haemopoietic cell lines by in situ hybridization and Northern Blot analysis using a radioisotope-labelled monkey Epo DNA probe. A positive message was expressed by a human cell line, CM-S, derived from a patient with congenital hypoplastic anemia, and by a murine erythro-leukaemic cell line, clone 707, derived from the spleen of Friend virus-infected mice. No message was detected in two megakaryoblastic cell lines, and in a monocytic cell line, derived from a patient with acute monocytic leukaemia. These data may fit with the hypothesis of expression of Epo and other growth factors by haemopoietic cells through a mechanism of so-called autocrine secretion.