[Adult stem cells: their scientific interest and therapeutic future]. / Cellules souches adultes: intérêt scientifique et avenir thérapeutique.

Fascinating and provocative findings have shaken the stem cell field during these past years, which may be exploited in the future in cell replacement therapies. Continuous renewal of blood, skin, and gut cells, has long be attributed to stem cells, but it was more unexpected to identify cells that fulfil the requirements for stem-progenitor cells in many tissues with a slow turnover such as heart, kidney, muscle and brain. However, despite their lack of risk and immunological barrier, adult stem cells are yet of poor therapeutic value in many diseases, because they are available in scarce number, are poorly amplified, and loose potential with ageing, among many obstacles. Thus, the identification in adult, and more recently fetal tissues, of cells with a high proliferative capacity and multi-lineage differentiation potential has been wellcome, although their existence is still a matter of controversy. An alternative would be to activate stem cells in situ, by acting on components of the niche as recently exemplified in the hematopoetic system. Finally, as fiction meets reality, it may become possible to reprogram human adult cells in pluripotent ES cells-like, as recently demonstrated in mice.

Successful transduction of human multipotent, lymphoid (T, B, NK) and myeloid, and transplantable CD34+CD38low cord blood cells using a murine oncoretroviral vector.

Hematopoietic stem cells (HSC) are subject to great interest because of their medical importance and their biological properties. Therefore, the possibility of genetically modifying human HSC is a major concern in several inherited pathologies. In this study, we aimed to demonstrate that a murine oncoretroviral vector can transduce multipotential cord blood (CB) stem cells. Sorted CB CD34(+)CD38(low) cells were transduced with a Moloney-based MFG retroviral vector containing the coding sequence of the murine CD2 (mCD2). CD34(+)mCD2(+) cells were sorted by flow cytometry and cultured either in bulk or at one cell per well in culture conditions that allow differentiation along lymphoid (T, B, and NK) and myeloid (M) lineages. Phenotypic analysis of cells generated in culture showed that CD34(+)mCD2(+) cells could give rise to all lymphoid and myeloid progeny, indicating that the MFG/mCD2 vector had transduced progenitors of all tested lineages. Moreover, clonal cultures of 660 CD34(+)mCD2(+) cells showed that approximately 5% of these cells were able to generate both myeloid and lymphoid (B + NK) progenies; for 25% of them, this included the production of lymphoid T cells. We also demonstrate that transduced CD34(+)CD38(low) CB cells with lymphoid and myeloid potentials were capable of engraftment into the bone marrow (BM) of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice during several months. These results show that MFG retroviral vectors can transduce multipotent (T, B, NK, M) human hematopoietic progenitors with in vivo repopulating activity.

The human embryo, but not its yolk sac, generates lympho-myeloid stem cells: mapping multipotent hematopoietic cell fate in intraembryonic mesoderm.

We have traced emerging hematopoietic cells along human early ontogeny by culturing embryonic tissue rudiments in the presence of stromal cells that promote myeloid and B cell differentiation, and by assaying T cell potential in the NOD-SCID mouse thymus. Hematogenous potential was present inside the embryo as early as day 19 of development in the absence of detectable CD34+ hematopoietic cells, and spanned both lymphoid and myeloid lineages from day 24 in the splanchnopleural mesoderm and derived aorta where CD34+ progenitors appear at day 27. By contrast, hematopoietic cells arising in the third week yolk sac, as well as their progeny at later stages, were restricted to myelopoiesis and therefore are unlikely to contribute to definitive hematopoiesis in man.

Efficient ex vivo expansion of NOD/SCID-repopulating cells with lympho-myeloid potential in hematopoietic grafts of children with solid tumors.

INTRODUCTION: The ex vivo expansion of hematopoietic grafts could be an important therapeutic tool for accelerating hematopoietic recovery after administration of high-dose chemotherapy regimens. The fate of the long-term repopulating cells during the ex vivo manipulation of grafts is a critical issue and will ultimately define the clinical applicability of this technology to hematopoietic transplantation. MATERIALS AND METHODS: To study the effects of a clinically applicable ex vivo expansion protocol in the proliferative potential of the most primitive human hematopoietic cells, both LTC-IC and NOD/SCID-RC assays were used to determine LTC-IC and NOD/SCID-RC contents of hematopoietic grafts, both before and after expansion (SCF, IL-3, PEG-MGDF Flt3-L and 5% AB serum), in four children with non-hematological malignancies. RESULTS: The mean percentage of CD34+ cells after expansion was 16%. The numbers of nucleated cells increased 20-fold with a mean three-fold increase in the numbers of CD34+ cells during the expansion period. The CFC content of the samples showed a mean 11-fold increase (range: 5-17) after ex vivo expansion. The primitive hematopoietic stem cell content of the expanded cell fraction evaluated by LTC-IC assays was found to be increased in two patients out of three, with maintenance of the LTC-IC frequency in the third patient. The NOD/SCID-RC potential, evaluated in five experiments from four patients using 109 mice injected 5-6 weeks earlier with human hematopoietic cells, increased from a mean percentage of 36% (range: 7-75%) before expansion, to a mean percentage of 70% (range: 37-100%) after expansion (P < 0.00001). The frequency of NOD/SCID-RC calculated with pooled data from all patients was 1/80,000 at day 0 and 1/40,000 after seven days of culture. The full phenotypic analysis of human hematopoietic cells obtained in NOD/SCID mice injected with expanded cells showed the presence of significant numbers of CD34+, CD19+ and CD15+ cells, suggesting the persistent lympho-myeloid potential of the expanded hematopoietic cells. CONCLUSION: Our results suggest that efficient expansion of NOD/SCID-RC with lympho-myeloid potential can be achieved not only in cord blood or normal marrow as previously reported, but also in hematopoietic grafts obtained from children exposed to high-dose chemotherapy.

CD133+ cell selection is an alternative to CD34+ cell selection for ex vivo expansion of hematopoietic stem cells.

CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. This study compared the expansion capacities of CD133(+) and CD34(+) cells isolated from the same cord blood (CB) samples. After 14 days culture in stroma-free, serum-free medium in the presence of stem cell factor (SCF), Flt3-1, megakaryocyte growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF), the CD133(+) and CD34(+) fractions displayed comparable expansion of the myeloid compartment (CFC, LTC-IC, and E-LTC-IC). The expansion of CD133(+) CB cells was up to 1262-fold for total cells, 99-fold for CD34(+) cells, 109-fold for CD34(+) CD133(+) cells, 133-fold for CFU-GM, 14.5-fold for LTC-IC, and 7.5-fold for E-LTC-IC. Moreover, the expanded population was able to generate lymphoid B (CD19(+)), NK (CD56(+)), and T (CD4(+) CD8(+)) cells in liquid or fetal thymic organ cultures, while expression of the homing antigen CXCR4 was similar on expanded and nonexpanded CD133(+) or CD34(+) cells. Thus, the CD133(+) subset could be expanded in the same manner as the CD34(+) subset and conserved its multilineage capacity, which would support the relevance of CD133 for clinical hematopoietic selection.

Enhanced transgene expression in cord blood CD34(+)-derived hematopoietic cells, including developing T cells and NOD/SCID mouse repopulating cells, following transduction with modified trip lentiviral vectors.

The recent development of lentivirus-derived vectors is an important breakthrough in gene transfer technology because these vectors allow transduction of nondividing cells such as hematopoietic stem cells (HSC), due to an active nuclear import of reverse-transcribed vector DNA. We recently demonstrated that addition of the central DNA flap of HIV-1 to an HIV-derived lentiviral vector strikingly increases transduction of CD34(+) cells. We now describe improvements of the transduction protocol designed to preserve HSC properties and two modifications of the previously described TRIP-CMV vector. First, deletion of the enhancer/promoter of the 3' LTR in the TRIP-CMV vector resulted in a safer vector (TRIPDeltaU3-CMV) with conserved transduction efficiency and increased EGFP transgene expression. Second, the original internal CMV promoter was replaced with the promoter for the ubiquitously expressed elongation factor 1alpha (EF1alpha). This promoter substitution resulted in a significantly more homogeneous expression of the EGFP transgene in all hematopoietic cell types, including CD34(+)-derived T lymphocytes, in which the CMV promoter was inactive, and NOD/SCID mouse repopulating cells. We thus present here an HIV-derived lentiviral vector, TRIPDeltaU3-EF1alpha, which can very efficiently transduce human cord blood HSC and results in high long-term transgene expression in CD34(+)-derived T, B, NK, and myeloid hematopoietic cells.

Different expression of CD41 on human lymphoid and myeloid progenitors from adults and neonates.

The glycoprotein (Gp) IIb/IIIa integrin, also called CD41, is the platelet receptor for fibrinogen and several other extracellular matrix molecules. Recent evidence suggests that its expression is much wider in the hematopoietic system than was previously thought. To investigate the precise expression of the CD41 antigen during megakaryocyte (MK) differentiation, CD34(+) cells from cord blood and mobilized blood cells from adults were grown for 6 days in the presence of stem cell factor and thrombopoietin. Two different pathways of differentiation were observed: one in the adult and one in the neonate cells. In the neonate samples, early MK differentiation proceeded from CD34(+)CD41(-) through a CD34(-)CD41(+)CD42(-) stage of differentiation to more mature cells. In contrast, in the adult samples, CD41 and CD42 were co-expressed on a CD34(+) cell. The rare CD34(+)CD41(+)CD42(-) cell subset in neonates was not committed to MK differentiation but contained cells with all myeloid and lymphoid potentialities along with long-term culture initiating cells (LTC-ICs) and nonobese diabetic/severe combined immune-deficient repopulating cells. In the adult samples, the CD34(+)CD41(+)CD42(-) subset was enriched in MK progenitors, but also contained erythroid progenitors, rare myeloid progenitors, and some LTC-ICs. All together, these results demonstrate that the CD41 antigen is expressed at a low level on primitive hematopoietic cells with a myeloid and lymphoid potential and that its expression is ontogenically regulated, leading to marked differences in the surface antigenic properties of differentiating megakaryocytic cells from neonates and adults. (Blood. 2001;97:2023-2030)

Stromal cells retard the differentiation of CD34(+)CD38(low/neg) human primitive progenitors exposed to cytokines independent of their mitotic history.

Stem cell proliferation induced by potent cytokines usually leads to a loss of primitive potential through differentiation. In this study, the ability of cytokines and murine MS5 stromal cells to independently regulate the proliferation and long-term culture-initiating cell (LTC-IC) activity of primitive CD34(+)CD38(low/neg) human bone marrow cells was evaluated. To compare populations with identical proliferation histories, cells were labeled with carboxy fluorescein diacetate succinimidyl ester, and LTC-IC activity was assessed 4 days later in cells that had accomplished the same number of divisions with or without MS5 cells. MS5 cells counteracted dramatically the loss of LTC-IC activity observed in the presence of cytokines alone. Thus, in the presence of MS5 cells, means of 1233 (n = 5) and 355 (n = 9) LTC-IC-derived colony-forming cells (CFCs) were generated by 1000 cells that performed 3 and 4 divisions respectively, whereas 311 (n = 5) and 64 (n = 5) CFCs were generated by 1000 cells cultured without MS5 cells. Interestingly, MS5 cells had no detectable effect on the LTC-IC activity of cells that divided only twice in 4 days-1606 CFCs (n = 6) and 1993 (n = 6) CFCs, respectively, without and with MS5 cells-and a 48 additional hours of coculture were necessary to unmask changes in the LTC-IC activity mediated by stromal cells. These results indicate that cytokines and stroma-derived signals can regulate independently the proliferation and differentiation of primitive cells and that these stroma-derived extracellular factors act directly on their target cells.

The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells.

Gene transfer in human hematopoietic stem cells (HSCs) has great potential for both gene therapy and the understanding of hematopoiesis. As HSCs have extensive proliferative capacities, stable gene transfer should include genomic integration of the transgene. Lentiviral vectors are now preferred to oncoretroviral vectors especially because they integrate in nondividing cells such as HSCs, thereby avoiding the use of prolonged cytokine stimulation. Human immunodeficiency virus type-1 (HIV-1) has evolved a complex reverse transcription strategy including a central strand displacement event controlled in cis by the central polypurine tract (cPPT) and the central termination sequence (CTS). This creates, at the center of HIV-1 linear DNA molecules, a 99-nucleotide-long plus-strand overlap, the DNA flap, which acts as a cis-determinant of HIV-1 genome nuclear import. The reinsertion of the DNA flap sequence in an HIV-derived lentiviral vector promotes a striking increase of gene transduction efficiency in human CD34(+) hematopoietic cells, and the complementation of the nuclear import defect present in the parental vector accounts for this result. In a short ex vivo protocol, the flap-containing vector allows efficient transduction of the whole hierarchy of human HSCs including both slow-dividing or nondividing HSCs that have multiple lymphoid and myeloid potentials and primitive cells with long-term engraftment ability in nonobese diabetic/severe combined immunodeficiency mice (NOD/SCID).

Polydom: a secreted protein with pentraxin, complement control protein, epidermal growth factor and von Willebrand factor A domains.

To identify extracellular proteins with epidermal growth factor (EGF) domains that are potentially involved in the control of haemopoiesis, we performed degenerate reverse-transcriptase-mediated PCR on the murine bone-marrow stromal cell line MS-5 and isolated a new partial cDNA encoding EGF-like domains related to those in the Notch proteins. Cloning and sequencing of the full-length cDNA showed that it encoded a new extracellular multi-domain protein that we named polydom. This 387 kDa mosaic protein contained a signal peptide followed by a new association of eight different protein domains, including a pentraxin domain and a von Willebrand factor type A domain, ten EGF domains, and 34 complement control protein modules. The human polydom mRNA is strongly expressed in placenta, its expression in the other tissues being weak or undetectable. The particular multidomain structure of the encoded protein suggests an important biological role in cellular adhesion and/or in the immune system.

In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34(+) cord blood cells.

The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity. Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture. These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.

[Hematopoietic stem cells]. / Cellules souches hématopoïétiques.

Hematopoietic stem cells, which share with other stem cells of adult tissues the ability to maintain constant the number and diversity of differentiated mature cells throughout adult life offer a fabulous system to analyze mechanisms controlling cell proliferation and differentiation. Cytokines controlling the differentiation of intermediate progenitors into mature cells of the various lineages have been characterized and have been widely used, in vitro as in vivo, to increase the output of differentiated cells. In contrast, despite significant technological advances, molecular events associated with the stem cell decisions first to either self-renew or differentiate, and then to irreversibly commit to one of the lymphoid or of the myeloid pathways are still very badly understood. This is partly explained by the lack of reliable assays, particularly in humans, to assess stem cell activity, and by the difficulty to dissect the composition of molecular complexes regulating gene expression in these very rare cells. Despite these limitations, recent evidence suggests that there is some flexibility in the initial decisions of stem cells, and that extracellular factors may influence stem cell fate. If this is confirmed, it may then become possible to propose new therapeutic strategies based on the manipulation of stem cell properties.

Neuropilin-1 is expressed on bone marrow stromal cells: a novel interaction with hematopoietic cells?

In adult bone marrow, hematopoietic stem cells are found in close association with distinctive stromal cell elements. This association is necessary for maintenance of hematopoiesis, but the precise mechanisms underlying the cross-talk between stromal cells and hematopoietic stem cells are poorly understood. In this study, we used a bone marrow stromal cell line (MS-5) that is able to support human long-term hematopoiesis. This hematopoietic-promoting activity cannot be related to expression of known cytokines and is abolished by addition of hydrocortisone. Using a gene trap strategy that selects genes encoding transmembrane or secreted proteins expressed by MS-5 cells, we obtained several insertions that produced fusion proteins. In one clone, fusion protein activity was downregulated in the presence of hydrocortisone, and we show that insertion of the trap vector has occurred into the neuropilin-1 gene. Neuropilin-1 is expressed in MS-5 cells, in other hematopoietic-supporting cell lines, and in primary stromal cells but not in primitive hematopoietic cells. We show that neuropilin-1 acts as a functional cell-surface receptor in MS-5 cells. Two neuropilin-1 ligands, semaphorin III and VEGF 165, can bind to these cells, and the addition of VEGF 165 to MS-5 cells increases expression of 2 cytokines known to regulate early hematopoiesis, Tpo and Flt3-L. Finally, we show that stromal cells and immature hematopoietic cells express different neuropilin-1 ligands. We propose that neuropilin-1 may act as a novel receptor on stromal cells by mediating interactions between stroma and primitive hematopoietic cells.

Murine stromal cells counteract the loss of long-term culture-initiating cell potential induced by cytokines in CD34(+)CD38(low/neg) human bone marrow cells.

Evidence has been provided recently that shows that high concentrations of cytokines can fulfill functions previously attributed to stromal cells, such as promote the survival of, and led to a net increase in human primitive progenitors initiating long-term cultures in vitro (LTC-IC) or engrafting NOD-SCID (nonobese diabetic severe-combined immunodeficient) recipients in vivo. These data prompted us to re-evaluate whether stromal cells will further alter the properties of primitive progenitor cells exposed to cytokines. Single CD34(+)CD38(low) and CD38(neg) cells were incubated 10 days in serum-containing or serum-free medium in the presence or in the absence of murine marrow-derived stromal cells (MS-5). Recombinant human cytokines stem cell factor (SCF), pegylated-megakaryocyte growth and differentiation factor (PEG-MGDF), FLT3-L, Interleukin (IL)-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were systematically added at various concentrations (10 to 300 ng/mL). Cell proliferation and LTC-IC potential were evaluated in each clone after 10 days. A striking and consistent observation was the retention of a high LTC-IC potential in clones exposed to cytokines in the presence of stromal feeders, whereas clones exposed to cytokines alone in the absence of stromal feeders rapidly lost their LTC-IC potential as they proliferated. This was reflected both by the higher proportion of wells containing LTC-IC and by the high numbers of CFC produced after 5 weeks in clones grown with MS-5 during the first 10 days. We further showed by analyzing multiple replicates of a single clone at day 10 that MS-5 cells promoted a net increase in the LTC-IC compartment through self-renewal divisions. Interestingly, these primitive LTC-IC were equally distributed among small and large clones, as counted at day 10, indicating that active proliferation and loss of LTC-IC potential could be dissociated. These observations show that, in primitive cells, stromal cells counteract differentiation events triggered by cytokines and promoted self-renewal divisions. Furthermore, the almost identical distribution of the size of the clones with or without MS-5 suggests that proliferation and function of human primitive cells may be independently regulated by external signals, and that the former is primarily under the control of cytokines.

Identification of lymphomyeloid primitive progenitor cells in fresh human cord blood and in the marrow of nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice transplanted with human CD34(+) cord blood cells.

Transplantation of genetically marked donor cells in mice have unambiguously identified individual clones with full differentiative potential in all lymphoid and myeloid pathways. Such evidence has been lacking in humans because of limitations inherent to clonal stem cell assays. In this work, we used single cell cultures to show that human cord blood (CB) contains totipotent CD34(+) cells capable of T, B, natural killer, and granulocytic cell differentiation. Single CD34(+) CD19(-)Thy1(+) (or CD38(-)) cells from fresh CB were first induced to proliferate and their progeny separately studied in mouse fetal thymic organotypic cultures (FTOCs) and cocultures on murine stromal feeder layers. 10% of the clones individually analyzed produced CD19(+), CD56(+), and CD15(+) cells in stromal cocultures and CD4(+)CD8(+) T cells in FTOCs, identifying totipotent progenitor cells. Furthermore, we showed that totipotent clones with similar lymphomyeloid potential are detected in the bone marrow of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice transplanted 4 mo earlier with human CB CD34(+) cells. These results provide the first direct demonstration that human CB contains totipotent lymphomyeloid progenitors and transplantable CD34(+) cells with the ability to reconstitute, in the marrow of recipient mice, the hierarchy of hematopoietic compartments, including a compartment of functional totipotent cells. These experimental approaches can now be exploited to analyze mechanisms controlling the decisions of such primitive human progenitors and to design conditions for their ampification that can be helpful for therapeutic purposes.

Identification of human T-lymphoid progenitor cells in CD34+ CD38low and CD34+ CD38+ subsets of human cord blood and bone marrow cells using NOD-SCID fetal thymus organ cultures.

In contrast to myeloid and B-lymphoid differentiation, which take place in the marrow environment, development of T cells requires the presence of thymic stromal cells. We demonstrate in this study that human CD34+, CD34+ CD38+ and CD34+ CD38(low) cells from both cord blood and adult bone marrow reproducibly develop into CD4+ CD8+ T cells when introduced into NOD-SCID embryonic thymuses and further cultured in organotypic cultures. Such human/mouse FTOC fetal thymic organ culture) thus represents a reproducible and sensitive system to assess the T-cell potential of human primitive progenitor cells. The frequency of T-cell progenitors among cord-blood-derived CD34+ cells was estimated to be 1/500. Furthermore, the differentiation steps classically observed in human thymus were reproduced in NOD-SCID FTOC initiated with cord blood and human marrow CD34+ cells: immature human CD41(low) CD8- sCD3- TCR alphabeta- CD5+ CD1a+ T cells were mixed with CD4+ CD8+ cells and more mature CD4+ CD8- TCR alphabeta+ cells. However, in FTOC initiated with bone marrow T progenitors, <10% double-positive cells were observed, whereas this proportion increased to 50% when cord blood CD34+ cells were used, and most CD4+ cells were immature T cells. These differences may be explained by a lower frequency of T-cell progenitors in adult samples, but may also suggest differences in the thymic signals required by bone marrow versus cord blood T progenitors. Finally, since cytokine-stimulated CD34+ CD38(low) cells retained their ability to generate T cells, these FTOC assays will be of value to monitor, when combined with other biological assays, the influence of different expansion protocols on the potential of human stem cells.

Characterization of multiple isoforms of protein 4.1R expressed during erythroid terminal differentiation.

In erythrocytes, 80-kD protein 4.1R regulates critical membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4. 1, generated by alternative pre-mRNA splicing, are expressed during erythroid differentiation. Erythroid precursors use two splice acceptor sites at the 5' end of exon 2, thereby generating two populations of 4.1 RNA: one that includes an upstream AUG-1 in exon 2' and encodes high molecular weight isoforms, and another that skips AUG-1 in exon 2' and encodes 4.1 by initiation at a downstream AUG-2 in exon 4. To begin an analysis of the complex picture of protein 4.1R expression and function during erythropoiesis, we determined the number and primary structure of 4.1R isoforms expressed in erythroblasts. We used reverse-transcription polymerase chain reaction to amplify and clone full-length coding domains from the population of 4.1R cDNA containing AUG-1 and the population excluding AUG-1. We observed an impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive characterization of 4.1R primary structures in a single-cell lineage. 4.1R isoforms, transfected into COS-7 cells, distributed to the nucleus, cytoplasm, plasma membrane, and apparent centrosome. We confirmed previous studies showing that inclusion of exon 16 was essential for efficient nuclear localization. Unexpectedly, immunochemical analysis of COS-7 cells transfected with an isoform lacking both AUG-1 and AUG-2 documented that a previously unidentified downstream translation initiation codon located in exon 8 can regulate expression of 4.1R. We speculate that the repertoire of primary structure of 4.1R dictates its distinct binding partners and functions during erythropoiesis.

Retrovirus-mediated gene transfer into human CD34+38low primitive cells capable of reconstituting long-term cultures in vitro and nonobese diabetic-severe combined immunodeficiency mice in vivo.

Factors that may improve retroviral transduction of primitive human hematopoietic cells were studied using MFG-based vectors containing a LacZ gene and produced either by a murine (psi-Crip) or a human (Tasaf) cell line. Cord blood (CB) or bone marrow (BM) CD34+ cells were stimulated and transduced in the presence of three cytokines (interleukin 3 [IL-3], IL-6, and stem cell factor [SCF; c-Kit Ligand]). In the supernatant infection protocol, hematopoietic progenitor cells as measured by X-Gal staining of colony-forming unit cells (CFU-Cs) were transduced more effectively with Tasaf (20%) than with psi-Crip (8%). In contrast, there was no difference between these two cell lines in a coculture protocol. However, gene transfer into more primitive CD34+CD38- subsets and in LTC-IC-derived colonies was low. The use of a large number of cytokines including FLT3-L and PEG-rhMGDF increased the transduction efficiency into CD34+CD38(-)-derived CFU-Cs (35% by PCR) or LTC-ICs (10%). A virus pseudotyped with gibbon ape leukemia virus (GALV) envelope further improved gene transfer to 60 and 48% for LacZ+ CFU-C- and LTC-IC-derived colonies, respectively. These conditions of transduction allowed multilineage engraftment of primitive cord blood cells in NOD-SCID mice. Moreover, 10% (at least) of the human hematopoietic cells recovered from the marrow of these immunodeficient animals were transduced. These data suggest that the efficiency of transduction of human hematopoietic primitive cells can be significantly improved by judicious combinations of recombinant cytokines and high retroviral titers.