Giovanni Migliaccio

Cloning of human erythroid progenitors (BFU-E) in the absence of fetal bovine serum

We describe culture conditions which enabled us to clone early human erythroid progenitors (BFU‐E) in the absence of fetal bovine serum (FBS). Our medium, which is chemically fully defined, supports proliferation and differentiation of erythroid progenitors comparable to that in FBS‐supplemented cultures. Furthermore, it allows cloning of BFU‐E from all human haemopoietic tissues (adult marrow, embryonic liver and yolk sac) and adult or perinatal blood. This system should facilitate the investigation of human erythroid differentiation in vitro (e.g. cell‐cell interaction. mechanism of action of haemopoietins and inhibitors, regulation of Hb synthesis) as well as the mechanisms underlying myeloproliferative disorders. As an example, we have found that recombinant Ep shows, in this culture system, the same dose response as Ep from conventional sources.

Human embryonic hemopoiesis: control mechanisms underlying progenitor differentiation in vitro

In order to investigate differences in control mechanisms between embryonic and adult hemopoiesis, we have studied the sensitivity of human embryonic progenitors (5-8 weeks postconception) to either positive (erythropoietin (Ep), granulocyte-macrophage colony-stimulating factor (GM-CSF) and insulin-like growth factor 1 (IGF-1] or negative (tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma] in vitro regulators of adult hemopoietic differentiation. Growth stimulators were analyzed under serum-deprived conditions whereas growth inhibitors were investigated in serum-supplemented culture. Formation of granulocyte-macrophage colonies from embryonic progenitors was induced by GM-CSF but inhibited by TNF and IFN-gamma. Early erythroid progenitors resemble adult erythroid burst-forming cells (BFU-E) in their sensitivity to Ep and TNF but differ in their lack of response to GM-CSF or other adult sources of burst-promoting activity, and absence of inhibition by IFN-gamma. IGF-1 promoted erythroid burst formation in the absence of insulin, but did not have Ep-like activity. These data indicate that embryonic and adult erythroid progenitors differ at least in terms of in vitro sensitivity to GM-CSF and IFN-gamma and suggest that different cellular response to control signals may underlie the differences observed in vivo between embryonic and adult hemopoiesis.

Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells.

In gene transfer experiments including gene therapy studies, expression of the integrated transgenes in host cells often declines with time. The molecular basis of this phenomenon is not clearly understood. We have used the Green Fluorescent Protein (GFP) gene as both a selectable marker and a reporter to study long-term transgene integration and expression in K562 cells. Cells transfected with plasmids containing the GFP gene coupled to the HS2 or HS3 enhancer of the human beta-globin Locus Control Region (LCR) or the cytomegalovirus (CMV) enhancer were sorted by either fluorescence-activated-cell-sorting (FACS) alone or FACS combined with drug selection based on a co-integrated drug resistance gene. The two groups of selected cells were subsequently cultured for long periods up to 250 cell generations. Comparison of long-term GFP transgene integration and expression in these two groups of cells revealed that the K562 genome contains two types of transgene integration sites: i) abundant unstable sites that permit transcription but not long-term integration of the transgenes and thus eliminate the transgenes in 60-250 cell generations and ii) rare stable sites that permit both efficient transcription and long-term stable integration of the transgenes for at least 200 cell generations. Our results indicate that extinction of GFP expression with time is due at least in part to elimination of the gene from the host genome and not entirely to transcriptional silencing of the gene. However, long-term, stable expression of the transgene can be achieved in cells containing the transgene integrated into the rare, stable host sites.

Identification of Two New Synthetic Histone Deacetylase Inhibitors That Modulate Globin Gene Expression in Erythroid Cells from Healthy Donors and Patients with Thalassemia

We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of γ-globin and/or β-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the γ/(γ+β) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of γ-globin mRNA and the γ/(γ+β) ratio (both by 2-fold). Compound 24 increased by 3-fold the level of γ-globin and decreased by 2-fold that of β-globin mRNA, increasing the γ/(γ+β) ratio by 6-fold, and raising (by 50%) the cell HbF content. Both compounds raised the acetylation state of histone H4 in primary cells, an indication that their activity was mediated through HDAC inhibition. Compounds 9 and 24 were also tested as γ/(γ+β) mRNA inducers in erythroblasts obtained from patients with β0 thalassemia. Progenitor cells from patients with β0 thalassemia generated in vitro morphologically normal proerythroblasts that, unlike normal cells, failed to mature in the presence of EPO and expressed low β-globin levels but 10 times higher-than-normal levels of the α hemoglobin-stabilizing protein (AHSP) mRNA. Both compounds ameliorated the impaired in vitro maturation in β0 thalassemic erythroblasts, decreasing AHSP expression to normal levels. In the case of two patients (of five analyzed), the improved erythroblast maturation was associated with detectable increases in the γ/(γ+β) mRNA ratio. The low toxicity exerted by compounds 9 and 24 in all of the assays investigated suggests that these new HDAC inhibitors should be considered for personalized therapy of selected patients with β0 thalassemia.

Translational research on advanced therapies

Fostering translational research of advanced therapies has become a major priority of both scientific community and national governments. Advanced therapy medicinal products (ATMP) are a new medicinal product category comprising gene therapy and cell-based medicinal products as well as tissue engineered medicinal products. ATMP development opens novel avenues for therapeutic approaches in numerous diseases, including cancer and neurodegenerative and cardiovascular diseases. However, there are important bottlenecks for their development due to the complexity of the regulatory framework, the high costs and the needs for good manufacturing practice (GMP) facilities and new end-points for clinical experimentation. Thus, a strategic cooperation between different stakeholders (academia, industry and experts in regulatory issues) is strongly needed. Recently, a great importance has been given to research infrastructures dedicated to foster translational medicine of advanced therapies. Some ongoing European initiatives in this field are presented and their potential impact is discussed.

In vivo expansion of purified hematopoietic stem cells transplanted in nonablated W/Wv mice.

We have evaluated the in vivo amplification potential of purified murine hematopoietic stem cells, identified as Wheat Germ Agglutinin+ (WGA+), 15-1.1(-) , Rhodamine 123 Dull (Rho-dull) cells, by serial transplantation into stem cell defective nonmyeloablated W/Wv mice. C57BL Rho-dull cells (250/ 500 cells/mouse) permanently engrafted nonablated W/Wv mice as defined by the presence of > 95% red and > 20% white donor-derived circulating cells for at least 1.5 years following transplantation. At this time, approximately 61% of Rho-dull cells and all the Rho-bright progenitor and colony forming cells of the engrafted mice were found to be donor-derived by c-Kit genotyping and by their response to stem cell factor (SCF). Retransplantation of 250-1000 Rho-dull cells from primary into secondary W/Wv recipients generated C57BL hematopoiesis in 40%-64% of animals revealing the presence of donor derived hematopoietic stem cells (HSC) in the bone marrow of the primary recipients. One and half years after transplantation, the bone marrow of the secondary engrafted animals contained C57BL Rho-dull cells approximately = 51% by genotype), which were capable of reconstituting tertiary W/Wv recipients. In this respect, 25% of tertiary mice expressed C57BL hematopoiesis when transplanted with 250-1000 Rhodull cells purified from secondary W/Wv recipients. On the basis of the number of Rho-dull cells purified from a single mouse, we calculate that approximately 7.3x10(4) Rho-dull cells, which are genotypically and functionally defined as C57BL long-term repopulating stem cells, were generated in the marrow of reconstituted primary W/Wv recipients transplanted 1.5 years earlier with 250-500 C57BL Rho-dull cells. We conclude that murine HSC have extensive amplification capacity in nonmyeloablated animals.

Growth factor receptor expression during in vitro differentiation of partially purified populations containing murine stem cells.

We have investigated, by semiquantitative RT‐PCR, the kinetics of activation of hematopoietic receptors and differentiation markers in partially purified murine hematopoietic stem cells (HSC) induced to differentiate in serum‐free culture with combinations of growth factor (GF). The combinations of GF used sustained either multilineage [stem cell factor (SCF) + interleukin 3 (IL‐3)], or erythroid [SCF + IL‐3 + erythropoietin (Epo)] or myeloid [SCF + IL‐3 + granulocyte colony‐stimulating factor (G‐CSF)] differentiation. The GF receptor genes investigated were the α and β subunits of the IL‐3 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) receptor, the erythropoietin receptor, the G‐CSF receptor, and c‐Fms, the receptor for macrophage colony‐stimulating factor (M‐CSF). The expression of Gata1 and α‐ and β‐globin was investigated at the same time as a marker of erythroid differentiation. HSC were purified according to standard protocols, which include partitioning of lineage‐negative bone marrow cells with the mitochondrial dye Rhodamine 123 (Rho) into Rho‐dull (≥17% of which reconstitute long‐term hematopoiesis in recipient mice) and into Rho‐bright (which are as capable as Rho‐dull of multilineage differentiation but do not permanently reconstitute the host). The following pattern of expression was observed: the α subunit of the IL‐3 receptor clearly was expressed in both Rho‐bright and Rho‐dull cells at the outset, and its expression did not change over time in culture. The β subunits of the IL‐3 and GM‐CSF receptor, the α subunit of the GM‐CSF receptor, the Epo and G‐CSF receptors and Fms barely were expressed in purified Rho‐bright and Rho‐dull cells, but their expression increased in cells cultured both in erythroid and in myeloid GF combinations. Gata1 was expressed maximally in Rho‐bright cells but was below the level of detection in Rho‐dull cells. Rho‐dull cells expressed Gata1 when cultured both in erythroid and in myeloid GF combinations. In contrast, α‐ and β‐globin, which also were not expressed in the purified cells, were induced only in cells stimulated with Epo. These results indicate that the genes for all the GF receptors investigated (with the exception of the α subunit of the IL‐3 receptor) are expressed at low levels, if any, in purified Rho‐bright or Rho‐dull cells, but are expressed in their progeny cultured either in erythroid or myeloid GF combinations. The expression of the Epo receptor,in particular, is activated both in erythroid (α‐ and β‐globin positive) and in myeloid (α‐ and β‐globin negative) cells. Therefore, activation of the expression of the Epo receptor gene and activation of the erythroid differentiation program are two independent events in normal hematopoiesis. J. Cell. Physiol. 171:343–356, 1997.

Stem cell factor induces proliferation and differentiation of fetal progenitor cells in the mouse

We have investigated the kinetics of the amplification of the progenitor cell compartments (CFC) in haemopoietic organs during murine ontogenesis and compared the growth requirements of fetal and adult CFC. Two haemopoietic phases were recognized in the fetal liver (FL): an exponential growth phase, from 11.5 to 15.5 d post conception (p.c.), during which the mean number of nucleated cells and of CFC in the FL increased from 4.9 × 105 to 7.0 × 107 and from 4.5 × 103 to 2.7 × 105, respectively, and a recessive phase after 15.5 d p.c., during which the CFC number in the FL gradually decreased, although some CFC were still detectable in the liver after birth. In serum‐deprived cultures, FL and adult marrow (AM) CFC had similar responses to GM‐CSF, and did not respond to G‐CSF or IL‐3. In contrast, FL, but not AM, erythroid colonies grew Epo‐independently whereas SCF alone induced formation of maximal numbers of erythroid bursts from FL, but not from AM cells. The proliferative and differentiative effect of SCF alone on fetal cells was confirmed in serum‐deprived cultures of purified early progenitor cells isolated by cell sorting on the basis of multiple parameters from FL and AM light‐density cells. In culture of purified FL cells, SCF alone induced a similar amplification of total cells (maximal amplification at day 12: 800–300‐fold) and total CFC (11–38‐fold of maximal amplification at day 6) to the combination of SCF plus IL‐3 (1300–800‐fold amplification of total cells and 31–88‐fold amplification of CFC). In contrast, SCF alone allowed only survival of purified AM early progenitor cells. Therefore FL early progenitor cells have an intrinsic higher potential than their adult counterpart to respond to SCF, confirming the potent role of this growth factor in the development of the murine haemopoietic system.

LONG-TERM GENERATION OF COLONY FORMING CELLS (CFC) FROM CD34+ HUMAN UMBILICAL CORD BLOOD CELLS

Human umbilical cord blood cells represent a potential alternative to bone marrow as a source of stem and progenitor cells for allogeneic transplantation. Therefore, many studies are underway to evaluate the number of cord blood stem cells and their amplification potential. We analyze here the amplification potential of CD34+ cord blood cells in liquid cultures stimulated with stem cell factor (SCF) in combination with interleukin-3 (IL-3), erythropoietin (Epo) or granulocyte colony-stimulating factor (G-CSF) under serum-deprived conditions. We report that under certain circumstances (stimulation with SCF and IL-3, replacing of the medium and growth factors every 3-4 days, no change of the initial culture flask, 37 degrees C as incubation temperature), CD34+ cells give rise to differentiated cells and progenitor cells for more than two months. During this period, more than 10(10) differentiated cells and 10(6) progenitor cells are generated from 0.25-1 x 10(4) CD34+ cells in the absence of a stromal layer. These data highlight the high proliferative and differentiative potential of cord blood stem cells and, because the culture procedures are relatively simple and do not require a stromal layer, open the way to the clinical use of ex vivo stem cell expansion.

Differential Amplification of Murine Bipotent Megakaryocytic/Erythroid Progenitor and Precursor Cells During Recovery from Acute and Chronic Erythroid Stress

Two murine bipotent erythroid/megakaryocytic cells, the progenitor (MEP) and precursor (PEM) cells, recently have been identified on the basis of the phenotypes of linnegc‐kitposSca‐1neg CD16/CD32lowCD34low and TER119pos4A5pos or 2D5pos, respectively. However, the functional relationship between these two subpopulations and their placement in the hemopoietic hierarchy is incompletely understood. We compared the biological properties of these subpopulations in marrow and spleen of mice with and without acute or chronic erythroid stress. MEP cells, but not PEM cells, express c‐kit, respond to stem cell factor in vitro, and form spleen colonies in vivo. PEM cells comprise up to 50%–70% of the cells in BFU‐E–derived colonies but are not present among the progeny of purified MEP cells cultured under erythroid and megakaryocytic permissive conditions. PEM cells increase 10‐ to 20‐fold under acute and chronic stress, whereas MEP cell increases (21%–84%) are observed only in acutely stressed animals. These data suggest that MEP and PEM cells represent distinct cell populations that may exist in an upstream‐downstream differentiation relationship under conditions of stress. Whereas the dynamics of both populations are altered by stress induction, the differential response to acute and chronic stress suggests different regulatory mechanisms. A model describing the relationship between MEP, PEM, and common myeloid progenitor cells is presented.