Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D.

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.

Human embryonic hemopoiesis. Kinetics of progenitors and precursors underlying the yolk sac----liver transition.

Human embryonic development involves transition from yolk sac (YS) to liver (L) hemopoiesis. We report the identification of pluripotent, erythroid, and granulo-macrophage progenitors in YS, L, and blood from human embryos. Furthermore, comprehensive studies are presented on the number of hemopoietic progenitors and precursors, as well as of other cell types, in YS, L, and blood at precisely sequential stages in embryos and early fetuses (i.e., at 4.5-8 wk and 9-10 wk postconception, respectively). Our results provide circumstantial support to a monoclonal hypothesis for human embryonic hemopoiesis, based on migration of stem and early progenitor cells from a generation site (YS) to a colonization site (L) via circulating blood. The YS----L transition is associated with development of the differentiation program in proliferating stem cells: their erythroid progeny shows, therefore, parallel switches of multiple parameters, e.g., morphology (megaloblasts----macrocytes) and globin expression (zeta----alpha, epsilon----gamma).

The thrombopoietin/MPL axis is activated in the Gata1low mouse model of myelofibrosis and is associated with a defective RPS14 signature.

Myelofibrosis (MF) is characterized by hyperactivation of thrombopoietin (TPO) signaling, which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. As mice carrying the hypomorphic Gata1low mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop MF, we investigated whether the TPO axis is hyperactive in this model. Gata1low mice contained two times more Tpo mRNA in liver and TPO in plasma than wild-type littermates. Furthermore, Gata1low LSKs expressed levels of Mpl mRNA (five times greater than normal) and protein (two times lower than normal) similar to those expressed by LSKs from TPO-treated wild-type mice. Gata1low marrow and spleen contained more JAK2/STAT5 than wild-type tissues, an indication that these organs were reach of TPO-responsive cells. Moreover, treatment of Gata1low mice with the JAK inhibitor ruxolitinib reduced their splenomegaly. Also in Gata1low mice activation of the TPO/MPL axis was associated with a RSP14 deficiency and a discordant microarray ribosome signature (reduced RPS24, RPS26 and SBDS expression). Finally, electron microscopy revealed that Gata1low megakaryocytes contained poorly developed endoplasmic reticulum with rare polysomes. In summary, Gata1low mice are a bona fide model of MF, which recapitulates the hyperactivation of the TPO/MPL/JAK2 axis observed in megakaryocytes from myelofibrotic patients.

Proliferation of purified murine hemopoietic stem cells in serum-free cultures stimulated with purified stem-cell-activating factor.

Under conditions of steady-state hemopoiesis in normal mice, the majority of hemopoietic stem cells in the bone marrow are in the quiescent state of the cell cycle. These cells can be stimulated to proliferate in vitro by the addition of a factor termed the "stem-cell-activating factor" (SAF), which is present in medium conditioned by various cell types. This factor is indistinguishable in antigenic and molecular properties from the lymphokine interleukin 3 (IL-3). The action of SAF on the stem cell cycle was studied by examination of the survival of the spleen colony-forming unit(s) (CFU-S) after four days of serum-free culture in the presence of purified SAF. CFU-S subtypes were distinguished on the basis of the day of colony counting (i.e., days 7, 9, and 12 after transplantation). The results indicate that SAF selectively induces an increase of the day-7 CFU-S: the CFU-S number increased 2.7-fold on day 7 and 1.2-fold on day 9, and decreased fivefold for day-12 CFU-S. Similar results were obtained in SAF culture of partially and highly purified stem cells. The proliferation of day-9 CFU-S in cultures of low-density bone marrow cells was found to be similar to that of unfractionated bone marrow cells until day 4 of culture. However, in the culture of partially purified stem cells, this proliferation stopped between days 4 and 10, whereas it continued with unfractionated cells. This indicates that cocultured bone marrow cells affect the proliferation of stem cells upon induction by SAF; day-4 cultures of highly purified resting stem cells with purified SAF resulted in a similar decrease in day-12 CFU-S and an increase in day-7 CFU-S, as was observed with unfractionated bone marrow cells. The DNA histogram of the stimulated sorted cells clearly revealed an actively DNA-synthesizing population. The results are in agreement with those of a selective induction of proliferation by SAF of resting pluripotent hemopoietic stem cells.

In vitro differentiation and proliferation of human hematopoietic progenitors: the effects of interleukins 1 and 6 are indirectly mediated by production of granulocyte-macrophage colony-stimulating factor and interleukin 3.

The effect of recombinant human interleukin (IL)-1 and IL-6 on the differentiation and proliferation in vitro of human granulocyte-macrophage (GM) and erythroid progenitors has been investigated in either fetal bovine serum (FBS)-supplemented or FBS-deprived cultures. Sources of progenitor cells were unfractionated bone marrow cells or marrow cells depleted of adherent and/or T cells. Each interleukin was investigated either alone or in combination with GM-colony-stimulating factor (CSF), IL-3 and erythropoietin (Epo), or granulocyte (G)-CSF. In FBS-supplemented cultures of unfractionated marrow cells, IL-1 induced optimal GM colony growth and increased by 50% the number of erythroid bursts that formed in the presence of Epo. The addition to these cultures of a neutralizing anti-GM-CSF monoclonal antibody or of an anti-IL-3 serum decreased the growth of GM colonies by 80% and 40%, respectively. Under the same conditions, IL-6 had no effect on GM colony growth but increased by 90% the number of erythroid bursts. This effect was partially (40%) neutralized by addition of anti-IL-3 serum. IL-1 and IL-6 were weak stimuli, or had no effect at all, either alone or in combination with GM-CSF and IL-3 in FBS-deprived cultures or in FBS-supplemented cultures of nonadherent or nonadherent, T-cell-depleted marrow cells. IL-1 and IL-6 had no effect, either alone or in combination with IL-3, in maintaining the number of progenitor cells in short-term liquid suspension cultures. These results indicate that the actions of IL-1 and IL-6 on hematopoiesis are mainly indirect and mediated by the production of GM-CSF and/or IL-3 by accessory cells. However, neither IL-1 nor IL-6 alone is sufficient to stimulate production of growth factor(s) by accessory cells, and at least a second stimulus, provided by FBS, is also required. These data are in agreement with a multisignal model of regulation of the expression of growth factor genes.

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.

Regulation of differentiation of murine progenitor cells derived from blast cell colonies under serum-deprived conditions.

We have examined the effect of interleukin 3 (IL-3), granulocyte-macrophage (GM)-, granulocyte (G)-, and macrophage (M)-colony-stimulating factors (CSFs) on the induction of GM colonies from highly enriched murine hematopoietic progenitor cells under serum-deprived conditions. Each growth factor was tested alone or in combination with suboptimal concentrations of the others. The effect of each CSF on GM colony growth in fetal bovine serum (FBS)-supplemented cultures of unfractionated marrow cells is reported for comparison. GM-CSF induced GM colony growth in serum-deprived cultures of purified progenitor cells to the same extent as in FBS-supplemented cultures of unfractionated marrow cells. In contrast, IL-3 was only one-tenth as active in promoting the growth of enriched progenitor cells under serum-deprived conditions when compared with its effect on colony growth from unfractionated marrow. M-CSF and G-CSF were almost completely ineffective in both cases. G-CSF induction of GM colony growth from purified progenitor cells was restored by addition of suboptimal concentrations of IL-3 or GM-CSF, suggesting that either IL-3 or GM-CSF is required to observe the effect of G-CSF. Addition of G-CSF to GM-CSF-stimulated cultures did not increase the maximal number of colonies detected, indicating that these two growth factors may act on the same subset of progenitor cells. Addition of GM-CSF or IL-3 to IL-3- or GM-CSF-stimulated cultures, respectively, increased by 40% the maximal number of colonies detected, suggesting that these two factors act on at least partially separate subsets of GM progenitors. These data parallel the recent observations on the control of human GM colony formation under FBS-deprived conditions and support a model for the control of myeloid differentiation that requires the interplay of different growth factors.

Dependence for the proliferative response to erythropoietin on an established erythroid differentiation program in a human hematopoietic cell line, UT-7.

Erythroid differentiation involves the activation of a number of erythroid-specific genes, most of which, including the globin genes and the erythropoietin receptor (Epo-R) gene, are, at least in part, regulated by the transcription factor GATA-1. In order to understand the relationship, if any, between expression of GATA-1, response to Epo and erythroid differentiation, we analyzed the expression of GATA-1, Epo-R and globin genes in an Epo-dependent human cell line, UT-7 Epo. The results were compared to those obtained with the parental granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell line, UT-7, which has a predominantly megakaryoblastic phenotype and is unable to proliferate continuously in the presence of Epo. UT-7 Epo and UT-7 expressed similar levels of GATA-1 mRNA and binding activity. The two lines also expressed comparable levels of Epo-R mRNA while the number of Epo-binding sites on UT-7 Epo cells was one-sixth the number of UT-7 cells (2400 +/- 3 vs. 13,800 +/- 300). This difference in the number of binding sites could be due to differences in cell surface (UT-7 cells are 20% smaller than the parental UT-7 cells) or in receptor turnover. By Northern analysis, UT-7 cells expressed detectable levels of beta- and gamma-globin but not alpha-globin. In comparison, UT-7 Epo cells expressed alpha-globin and higher levels of gamma-globin (5-fold) and beta-globin (from barely to clearly detectable). Globin chains (alpha, beta and gamma) were clearly detectable by affinity chromatography in UT-7 Epo but not in UT-7 cells. The frequency of the cells which expressed beta- and gamma-globin genes in the two cell populations was measured by immunofluorescence with beta- and gamma-specific antibodies. The number of gamma-positive cells and their fluorescence intensity were higher in UT-7 Epo than in UT-7 cells (0 to 17% barely positive cells and 23 to 40% clearly positive cells, respectively), indicating that the increase in globin mRNA observed in UT-7 Epo is due to both an increase of gene expression per cell and an increase in numbers of cells containing gamma-globin. The levels of GATA-1, Epo-R and globin mRNA expressed were not affected by a 24-hour incubation of either cell line with Epo, GM-CSF or interleukin-3 (IL-3).(ABSTRACT TRUNCATED AT 400 WORDS)

Erythropoietic differentiation in humans: in vitro studies on erythroid progenitors and Hb synthesis in fetal, newborn and adult life.

Human erythroid progenitors from fetal liver, cord or adult blood and adult marrow were cultured in methylcellulose, according to standard techniques. Their clonogenetic features (colony morphology and number, time/growth curve, erythropoietin (Ep) and burst-enhancing factor (BEF) sensitivity, in vitro 3H-thymidine suicide index, etc) were comparatively investigated. Three classes of fetal liver erythroid progenitors (primitive or intermediate BFU-E, CFU-E) have been thereby identified and characterized. Furthermore, globin chains (alpha, beta, G gamma, A gamma) synthesis has been evaluated in single erythroid colonies, either well-or poorly-hemoglobinized, by means of a novel technique including analytical iso-electric focusing (IEF), sometimes preceded by preparative IEF separation of HbF and HbA. On the basis of these results, a model for the regulation of Hb synthesis is proposed here.

Alternatively spliced mRNAs encoding soluble isoforms of the erythropoietin receptor in murine cell lines and bone marrow.

32D Epo and 32D GM cells are subclones of the murine 32D cell line which are selectively dependent for proliferation and survival on erythropoietin (Epo) or granulocyte/macrophage colony-stimulating factor (GM-CSF), respectively. 32D GM cells were previously shown to express significant levels of the Epo receptor mRNA and protein which was retained intracellularly and did not appear on the cell surface. We have now analyzed the EpoR mRNA from the 32D GM line, using PCR followed by direct sequencing. Several alternatively spliced products were detected. In some molecules, intron 5 (I5) or part of I6 or both were retained. Retention of I5 results in a mRNA potentially encoding an almost complete extracellular domain, while retention of I6 gives rise to a mRNA encoding the complete extracellular and transmembrane domains. A different type of splicing results in the loss of exon 5 (E5), giving rise to a sequence encoding a truncated extracellular domain. These alternatively spliced sequences are differentially represented in 32D Epo versus 32D GM cells. All are additionally present in normal bone marrow cells. Apart from these alternatively spliced EpoR RNAs, no other abnormalities were detected in EpoR RNA from 32D GM cells that could account for the intracellular retention of EpoR in the non-erythroid subclones of 32D.

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.

Amplification of T cells from human cord blood in serum-deprived culture stimulated with stem cell factor, interleukin-7 and interleukin-2.

We report the effects exerted by cytokine combinations, including stem cell factor (SCF), interleukin-7, interleukin-4 and interleukin-2, on the amplification of T cells from cord blood (CB) mononuclear cells cultured for 10-11 days under serum-deprived conditions. Of all the combinations investigated, SCF+interleukin-7 sustained the best fold increase (FI) of total nucleated cells (FI=6.4+/-1.17), amplifying preferentially CD4(+) over CD8(+) T-cell subsets (FI=4.72+/-0.79 vs 2.73+/-1.2, respectively, P<0.05). The addition of interleukin-2 to this combination did not significantly increase the total number of cells generated (FI=7.4+/-2.27), but allowed preferential amplification of CD8(+) over CD4(+) T cells (FI=6.04+/-0.14 vs 1.67+/-0.6, respectively, P<0.05). Single-strand conformation polymorphism analysis of the T-cell receptor V(beta)-chain rearrangements expressed by the expanded T cells indicated that the complexity of the T-cell repertoire had increased after 10 days of culture in the presence of SCF and IL-7. Interestingly, a modest expansion (FI=8.67+/-1.5) of myeloid progenitor cells was also observed in these cultures. These results indicate that it is possible to expand specific T-cell subsets for adoptive immunotherapy without losing myeloid progenitor cells necessary for neutrophil recovery after CB transplantation, by modulating the cytokines added to the cultures.

Circulating hematopoietic progenitor cells in a fetus with alpha thalassemia: comparison with the cells circulating in normal and non-thalassemic anemia fetuses and implications for in utero transplantations.

Our aim was to evaluate the number of progenitor cells circulating in an alpha-thalassemic fetus during its infusion in utero with paternal CD34(+) and adult red cells and to compare those values with those circulating in normal and non-thalassemic anemic fetuses of matched gestational age. The treatment of the alpha-thalassemic fetus has been described elsewhere. Fetal blood was obtained from normal and anemic fetuses by fetal blood sampling for diagnostic or therapeutic purposes according to a protocol approved by the human subject committee. The number of progenitor cells in fetal blood was estimated on the basis of the number of colonies they gave rise to in semisolid cultures. The alpha-thalassemic fetus, as did the other fetuses analyzed, contained high numbers (10(6)-10(7) depending on the age) of progenitor cells, values which were higher than the number (10(4)-10(5)) of paternal progenitor cells being transplanted. Progenitor cells with adult characteristics (adult kinetics of differentiation) were detected rapidly (10 min) after the CD34(+) cell infusion, but were not detectable 2-3 weeks after the transplant. These results indicate that adult progenitor cells do not have a numerical advantage when transplanted into alpha-thalassemic fetuses.

Characterization of the T cell receptor repertoire of neonatal T cells by RT-PCR and single strand conformation polymorphism analysis.

We have analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) the individual non-germ line configurations of the T cell receptor (TCR) Vbeta chains expressed by T cells from eight individual cord blood specimens. cDNA from each cord blood was amplified using a common primer coupled with a primer specific for each of 22 variable elements of the Vbeta chain family and the amplified fragments were separated under high resolution conditions. With cDNA from adult blood (as a control), all of the TCR chains were amplified as a smear consistent with the extensive polyclonality of adult T cells. In contrast, a heterogeneous pattern of amplification was observed with cDNAs from cord blood: only 26.7+/-21.9% of the 22 Vbeta chains analyzed were amplified as a smear. The majority of them were amplified as a discrete number of bands (up to 10) (in 68.2 +/-18.7% of samples) and some of them as a single fragment (4.0+/-7.8%). Only one of the eight samples analyzed expressed the majority (72.7%) of its Vbeta chains as a smear, consistent with an adult-like TCR repertoire. In conclusion, cord blood expressed, on average, a less complex TCR repertoire than adult blood.

The effect of different thawing methods, growth factor combinations and media on the ex vivo expansion of umbilical cord blood primitive and committed progenitors.

Assuming a threshold of 2 x 10(7) nucleated cells (NC)/kg body weight required for transplantation and 10 +/- 5 x 10(8) NC per cord blood (CB) unit (n = 1828, July 1997), 100%, 65% and 25% of the CB units stored in the CB Bank Düsseldorf contain sufficient NC to engraft patients of 10 kg, 35 kg and 50-70 kg, respectively. Thus, there is a potential limitation for the use of CB in adults which, however, may be overcome by ex vivo expansion of cells important in the different phases of engraftment. Therefore, four combinations of SCF, Flt3-L, IL-3, erythropoietin and GM-CSF as well as three media were evaluated for their capacity to amplify hematopoietic progenitors. A prerequisite for expansion was the significantly higher recovery of CD34+ cells, colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC) by thawing cryopreserved CB units with an isotonic albumin/dextran solution. When CD34+ CB cells were cultured with the four cytokine combinations in H5100 medium, all combinations promoted an expansion of total cells (43 to 356-fold) and CFC (49 to 462-fold) within 7 days, however, early progenitors as defined by mixed-colony formation (CFU-GEMM) were substantially amplified only with SCF, Flt3-L plus IL-3 (94.3 +/- 62.4-fold). H5100 medium or a serum-free medium supplemented with SCF, Flt3-L plus IL-3 were superior to 20% FCS/RPMI-1640 medium in the expansion of all progenitor cell types and were similarly effective in supporting the amplification of total cells, CFC, CFU-GM, BFU-E/CFU-E and LTC-IC (maximum at day 7: 6.7 +/- 3.4-fold and 5.5 +/- 0.5-fold, respectively). However, the serum-free medium promoted a significantly higher expansion of CFU-GEMM (176.9 +/- 81.7-fold) than H5100 medium (83.5 +/- 26.2-fold) at day 7 and only under serum-free conditions, CFU-GEMM were maintained over 14 days in tissue culture. These results demonstrate that committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be substantially amplified at the same time without exhausting the proliferative potential.

Early hemopoietic differentiation: the action of multi-CSF is complemented by lineage specific growth factors.

Although mechanisms controlling differentiation of hemopoietic stem and early progenitor cells are still poorly understood, it is generally conceded that a pivotal role is played by hemopoietic growth factors (HGFs). However, in-vitro analysis of their action on early progenitors may be obscured by cell-cell interaction, as well as by the presence of fetal bovine serum (FBS). To overcome these limitations, we investigated the action of pure multipotent or lineage-specific HGFs on purified progenitors grown in FBS-free cultures. In the murine system, highly purified progenitors were cultured in the presence of multipotent colony-stimulating factor (multi-CSF, also termed interleukin-3), erythropoietin (Ep) and macrophagic-CSF (M-CSF). Each HGF was unable by itself to induce significant colony growth. However, combined addition of multi-CSF and either Ep or M-CSF gave rise only to pure erythroid or macrophagic colonies, respectively. Partly purified human progenitors were challenged by human granulomonocytic-CSF (GM-CSF), pluripotent CSF (PPO, also termed granulocytic-CSF, G-CSF) and Ep. Here again, each HGF was unable per se to promote colony growth, but combined addition of GM-CSF or PPO and Ep gave rise only to pure erythroid colonies. These results support a model of early hemopoietic differentiation according to which multi-lineage HGFs represent "competence" GFs, the action of which is complemented by lineage-specific "progression" HGFs.

Molecular control of erythroid differentiation.

The number of circulating red cells is regulated by the daily balance between two processes: the destruction of the old red cells in the liver and the generation of new cells in the bone marrow. The process during which hematopoietic stem cells generate new red cells is called erythropoiesis. This article describes the most recent advances in molecular and cellular biology which have allowed the identification of the molecular mechanisms involved in the process of erythroid differentiation. It reviews the cellular compartments involved in the process, what is known on how these cells respond to erythroid specific growth factors and how the cells progressively activate specific transcription factors in order to express genes involved in the establishment of the erythroid phenotype.

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.

Standardization of progenitor cell assay for cord blood banking.

Cord blood has proved itself, if correctly stored with rational criteria, an excellent source of stem cells for related and unrelated transplants. It has been recently proven that the factor which predicts the best the speed of engraftment in cord blood transplants in the dose of progenitor cells injected per kg of body weight of the recipient. This result has been obtained thanks to a careful standardization of the neonatal progenitor cell assay. This manuscript describes such a standardization realized as a joined effort by the Istituto Superiore di Sanità, Rome, and the pivotal cord blood bank founded as a feasibility study by the National Institutes of Health, Bethesda at the New York Blood Center.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and leukemia inhibitory factor (LIF) protect the repopulating ability of purified murine hematopoietic stem cells in serum-deprived cultures stimulated with SCF and IL-3.

The engraftment potential of murine stem cells (HSC) is greatly reduced when these cells are expanded in vitro with stem cell factor and interleukin-3. We have evaluated if the addition of MIP-1 alpha or LIF to these cultures would protect the ability of murine wild type HSC to engraft the stem cell defective W/Wv recipient. In this transplantation model red and white blood cell reconstitution is assessed by hemoglobin electrophoresis and c-kit PCR genotyping, respectively. The results obtained indicate that both MIP-1 alpha and LIF protect, at least transiently, the HSC repopulating ability in vivo in spite of the modest expansion in the number of nucleated and progenitor cells observed.