Cytokine-induced selective expansion and maturation of erythroid versus myeloid progenitors from purified cord blood precursor cells

To study the role of different cytokine combinations on the proliferation and differentiation of highly purified primitive progenitor cells, a serum-free liquid culture system was used in combination with phenotypic and functional analysis of the cells produced in culture. CD34+ CD45RAlo CD71lo cells, purified from umbilical cord blood by flow cytometry and cell sorting, were selected for this study because of their high content of clonogenic cells (34%), particularly multipotent progenitors (CFU-MIX, 12% of all cells). Four cytokine combinations were tested: (1) mast cell growth factor (MGF; a c-kit ligand) and interleukin-6 (IL-6); (2) MGF, IL-6, IL-3, and erythropoietin (Epo); (3) MGF, IL-6, granulocyte-macrophage colony- stimulating factor (GM-CSF)/IL-3 fusion protein (FP), macrophage colony- stimulating factor (M-CSF), and granulocyte-CSF (G-CSF); and (4) MGF, IL-6, FP, M-CSF, G-CSF, and Epo. Maximum numbers of erythroid progenitors (BFU-E, up to 55-fold increase) and mature erythroid cells were observed in the presence of MGF, IL-6, IL-3, and Epo, whereas maximum levels of myeloid progenitors (CFU-C, up to 70-fold increase) and mature myeloid cells were found in cultures supplemented with MGF, IL-6, FP, M-CSF, and G-CSF. When MGF, IL-6, FP, M-CSF, G-CSF, and Epo were present, maximum levels of both erythroid and myeloid progenitors and their progeny were observed. These results indicate that specific cytokine combinations can act directly on primitive hematopoietic cells resulting in significant expansion of progenitor cell numbers and influencing their overall patterns of proliferation and differentiation. Furthermore, the observations presented in this study suggest that the cytokine combinations used were unable to bias lineage commitment of multipotent progenitors, but rather had a permissive effect on the development of lineage-restricted clonogenic cells.     

Characterization of functionally distinct subpopulations of CD34+ cord blood cells in serum-free long-term cultures supplemented with hematopoietic cytokines

We have previously identified, based on the expression of the CD45RA and CD71 antigens, three major subpopulations of CD34+ cells derived from human umbilical cord blood: CD34+ CD45RAloCD71lo cells (up to 42% multipotent progenitors), CD34+ CD45RA+ CD71lo cells (90% myeloid progenitors), and CD34+ CD45RAloCD71+ cells (70% erythroid progenitors). In the present study, we have investigated the long-term proliferation and differentiation of these subpopulations in response to hematopoietic cytokines. Cells from each subpopulation were cultured for 38 days in serum- and stroma-free liquid cultures supplemented with cytokine combinations that favor either erythropoiesis or myelopoiesis. In keeping with their high content of primitive progenitors, CD34+ CD45RAloCD71lo cells showed the highest CD34+ cell expansion (up to 532- fold) throughout the culture period, followed by CD34+ CD45RA+ CD71lo (130-fold) and CD34+ CD45RAloCD71+ (28-fold) cells. Interestingly, the cytokine combination favoring myelopoiesis was always more efficient in inducing CD34+ cell expansion than the one favoring erythropoiesis. In all but one of the cultures, a predominance of myelopoiesis was observed after 2 weeks, even in those supplemented with the cytokine mixture that favors erythropoiesis. Only when CD34+ CD45RAloCD71+ cells were cultured in the presence of erythroid cytokine mixture, erythropoiesis was evident at all time points. However, such cultures could be sustained for only 29 days. The results of this study demonstrate that the cord blood-derived CD34+ cell compartment consists of functionally distinct cell subpopulations that possess different proliferative capacities in vitro. Our results also show that the cytokine combinations used here were able to modulate proliferation and, to a much lesser extent, differentiation of such subpopulations, probably by favoring the expansion of committed progenitors rather than by acting on uncommitted cells.     

Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy- 1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200-10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony- forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.     

Expression of CD33, CD38, and HLA-DR on CD34+ human fetal liver progenitors with a high proliferative potential

High proliferative-potential colony-forming cells (HPP-CFC) have been identified in the bone marrow of mice and adult humans, and have been characterized as a compartment of primitive progenitors possibly including stem cells. In this report we describe the human fetal liver (FL) as a source of HPP-CFC. These FL HPP-CFC develop in clonal cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) within 3 to 4 weeks. The median frequency of HPP-CFC in FL tissues between 16 and 21 weeks of gestational age was 1 in 3,000 total FL cells. After 4 weeks of growth, FL HPP-CFC grew to a median colony size of 8.3 x 10(4) cells/colony. Using cell-sorting techniques FL HPP-CFC were shown to be predominantly contained in the CD34+ CD33+ CD38- fraction of FL cells. FL HPP-CFC were heterogeneous for HLA-DR expression, and no differences in proliferative capacities were observed between HLA-DR+ and HLA-DR- HPP- CFC. The CD34+ CD33-HLA-DR- CD38- population, previously suggested to contain stem cells, was observed to be very rare in the FL, representing approximately 1 in 1.7 x 10(5) light-density FL cells and containing almost no CFC. Therefore, it is possible that stem cells are contained in the CD33+ fraction of FL cells. Phenotypic characterization of CD34+ CD33+ CD38- lin -LDFL cells showed that these cells are also CD13+, predominantly Thy-1+, CD45RA-, CD45RO-, CD71-, and heterogenoeous for c-kit expression. These data suggest that FL HPP- CFC represent a heterogeneous compartment of primitive myeloid progenitors that may include stem cells.     

Allogeneic blood stem cell transplantation: peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34+ Thy- 1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease

Apheresis-derived hematopoietic progenitor cells have recently been used for allogeneic transplantation. Forty-one normal donors were studied to assess the effects of recombinant human granulocyte colony- stimulating factor (rhG-CSF) (12 micrograms/kg/d) on the peripheralization of hematopoietic progenitor cells and lymphoid subsets. The white blood cell, polymorphonuclear cell (PMNC), and lymphocyte concentrations at the peak of rhG-CSF effect in the donor's peripheral blood (PB) exceeded baseline by 6.4-, 8.0-, and 2.2-fold, respectively. Corresponding concentrations of PB CD34+ cells and primitive subsets such as CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells increased by 16.3-fold, 24.2-fold, and 23.2-fold, respectively in eight normal donors. The percentage of CD34+ Thy-1dim and CD34+ Thy- 1dim CD38- cells among CD34+ cells increased as well, suggesting an additional peripheralization effect of rhG-CSF on primitive CD34+ subsets. The preapheresis PB CD34+ and CD34+ Thy-1dim cell concentrations were predictive of their corresponding apheresis yield per liter of donor blood processed PB lymphoid subsets were not significantly affected by rhG-CSF treatment. The mean apheresis-derived yield of CD34+, CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells per kilogram of recipient body weight and per liter of donor blood processed was 48.9 x 10(4) (n = 41), 27.2 x 10(4) (n = 10), and 1.9 x 10(4) (n = 10), respectively. As compared with 43 single bone marrow (BM) harvest, the CD34+ cell yield of peripheral blood progenitor cell allografts of 41 normal donors exceeded that of BM allografts by 3.7- fold and that of lymphoid subsets by 16.1-fold (CD3+), 13.3-fold (CD4+), 27.4-fold (CD8+), 11.0-fold (CD19+), and 19.4-fold (CD56+CD3-). All PBPC allografts were cryopreserved before transplantation. The mean recovery of CD34+ cells after freezing, thawing, and washing out dimethylsulfoxide was 86.6% (n = 31) and the recovery of lymphoid subsets was 115.5% (CD3+), 121.4% (CD4+), 105.6% (CD8+), 118.1% (CD19+), and 102.4% (CD56+CD3-). All donors were related to patients: 39 sibling-to-sibling, 1 parent-to-child, and 1 child-to-parent transplant. Thirty-eight transplants were HLA fully identical, two transplants differed in one and two antigens. Engraftment occurred in 38 recipients; two patients died too early to be evaluated, and one patient did not engraft. The lowest CD34+ cell dose transplanted and resulting in complete and sustained engraftment was 2.5 x 10(6)/kg of recipient body weight.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.     

Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage-- (Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin- ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA- cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.     

Primitive human hematopoietic cells displaying differential efflux of the rhodamine 123 dye have distinct biological activities

Human bone marrow (BM) CD34+ cells were stained with the vital dye, rhodamine 123 (Rh123), and analyzed for their biological properties based on the level of dye retention. Heterogeneous rhodamine staining is seen within the CD34+ population, and the staining patterns differ dramatically between fetal BM (FBM), adult BM (ABM) and mobilized peripheral blood (MPB). Kinetic analysis of the efflux of Rh123 from ABM CD34+ cells showed that efflux of Rh123 was most rapid from the most primitive Thy-1+ subset. The efflux of Rh123 could be inhibited by verapamil, suggesting that rhodamine efflux from primitive hematopoietic cells is primarily due to the P-glycoprotein (P-gp) pump or another intracellular transport system affected by verapamil. When four CD34+ subpopulations were plated onto SyS1 BM stromal cell cocultures after 1 to 2 weeks, only wells plated with CD34+ Thy- 1+Rh123lo (low-level Rh123 retention) or CD34+Thy-1+Rh123mid (mid-level Rh123 retention) cells maintained greater than 50% of cells in an uncommitted CD34+33- stage. CD34+Lin- (lineage-negative) cells were fractionated based on Rh123 dye staining into Rh123hi (high-level Rh123 retention), Rh123mid, and Rh123lo and deposited as single cells into long-term SyS1 BM stromal cell cultures. The Rh123mid fraction had immense early proliferative activity in vitro, but lost the ability to form cobblestone areas after 5 to 6 weeks in culture. In contrast, the Rh123lo fraction proliferated more slowly but sustained long-term in vitro hematopoiesis as evidenced by continued cobblestone area-forming cells (CAFC) activity for at least 6 weeks. The Rh123hi fraction showed a plating efficiency similar to that of the Rh123lo or Rh1123mid fractions but did not extensively proliferative in vitro and did not show evidence of CAFC activity. We predicted from these in vitro results that the Rh123lo subsets possesses long-term engrafting potential. Indeed, on transplantation into the SCID-hu bone assay, all long-term engrafting potential and multilineage differentiation potential resided within the Rh123lo-mid but not Rh123hi subset. Furthermore, human marrow subpopulations derived from chimeric sheep after in utero transplantation with CD34+Thy-1+Lin- cells were reisolated based on Rh123 staining. Again, CD34+Lin- subsets showing Rh123lo-mid had long-term growth in culture, whereas Rh123hiCD34+Lin- cells did not. These results show that, after injection of CD34+Thy- 1+Lin- cells into an in utero microenvironment, primitive CD34+ cells maintain a Rh123 phenotype that correlates with their in vitro CAFC activity. Thus, Rh123 staining is an effective way to define functional subsets of primitive hematopoietic cell populations.     

Isolation of a candidate human hematopoietic stem-cell population.

We have identified a rare (0.05-0.1%) subset of human fetal bone marrow cells that contains multipotent hematopoietic precursors. The population of human precursor cells that express Thy-1 and CD34 but no known lineage markers is enriched for clonogenic activity that establishes long-term, multilineage (myelomonocytic and B lymphoid) cultures on mouse marrow stromal lines. Further, the Thy-1+CD34+ subset that takes up little of the fluorescent mitochondrial dye rhodamine 123 contains virtually all the cells that establish long-term cultures. In human fetal thymus transplanted into SCID (severe combined immunodeficiency) mice, Thy-1+CD34+ fetal bone marrow cells differentiate into T lymphocytes. In two of nine cases, allogeneic Thy-1+CD34+ cells could engraft intact human fetal bone marrow grown in SCID mice, resulting in donor-derived myeloid and B cells. By extrapolation, the rare human Thy-1+Lin-CD34+ cell population contains pluripotent hematopoietic progenitors; we propose that it is highly enriched for candidate hematopoietic stem cells.     

Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+ Thy-1+ cells into rapid division.

Various combinations of cytokines have profoundly different effects on inhibition of apoptosis and stimulation of self-renewal division of hematopoietic stem cells (HSC) in short-term, ex vivo culture. Our goal was to quantitate expansion of cells with a primitive CD34+ Thy-1+ phenotype, as well as cell cycling, division history, differentiation, and apoptosis of CD34+ cells enriched from normal donor mobilized peripheral blood (MPB) cells. The balance of these parameters determines the net number of transplantable HSC produced in ex vivo cultures. Comparing several different combinations of cytokines added to 90-hour cultures of MPB CD34 cells, thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL) gave the best result, with the lowest percentage of apoptotic cells and a mean 1.2-fold increase in the number of CD34+ Thy-1+ cells. A combination of interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) gave the worst outcome, including a decrease of CD34+ Thy-1+ cell number to a mean of 30% of the starting cell number. Cell division history was tracked using the dye 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE). Division of CD34+ Thy-1+ cells was faster and more synchronous in TPO, FL, and KL than in IL-3, IL-6, and LIF, which left a significant proportion of CD34+ cells undivided. Such detailed analyses of short-term, ex vivo cultures generated "replication scores," which allowed prediction of a sixfold improvement of the efficiency of gene transduction of primitive hematopoietic progenitors from MPB, using TPO, FL, and KL to replace IL-3, IL-6, and LIF. Analysis of retroviral transduction efficiency confirmed the increase of transgene expression from MPB primitive hematopoietic progenitors assayed after stromal culture was fivefold, validating the usefulness of multiparameter analysis of short-term cultures for survival and replication of CD34+ Thy-1+ cells.     

Blood-derived autografts collected during granulocyte colony- stimulating factor-enhanced recovery are enriched with early Thy-1+ hematopoietic progenitor cells

It was the objective of the study to characterize CD34+ hematopoietic progenitor cells from peripheral blood (PB) and bone marrow (BM) in a group of 24 cancer patients. After cytotoxic chemotherapy, R-metHu granulocyte colony-stimulating factor (R-metHuG-CSF; filgrastim, 300 micrograms daily, subcutaneously) was given to shorten the time of neutropenia as well as to increase the rebound of peripheral blood progenitor cells (PBPC) for harvesting. The proportion of CD34+ cells in the leukapheresis products (LPs) was 1.4-fold greater than in BM samples that were obtained at the same day (LP: median, 1.4% v BM: median, 1.0%, P < .01). Two- and three-color immunofluorescence showed that blood-derived CD34+ cells comprised a greater proportion of a particular early progenitor cell than CD34+ cells of bone marrow. Blood- derived progenitor cells tended to have a higher mean fluorescence intensity of CD34 and expressed significantly lower levels of HLA-DR (mean fluorescence intensity of HLA-DR: 442.6 +/- 44.9 [LP] v 661.5 +/- 64.6 [BM], mean +/- SEM, P < .01). Furthermore, the blood-derived CD34+ cells comprised a 1.7-fold greater proportion of Thy-1+ cells (LP: median, 24.4% v BM: median, 14.4%, P < .001) and expressed significantly less c-kit (LP: median, 20.5% v BM: median, 31.0%, P < .01). Three-color analysis showed that high levels of Thy-1 expression were restricted to CD34+/HLA-DRdim or CD34+/HLA-DR- cells confirming the early developmental stage of this progenitor cell subset. The proportion of CD34+/CD45RA(bright) cells representing late colony- forming unit granulocyte-macrophage (CFU-GM) was smaller in LPs compared with BM (P < .05). For an examination of BM CD34+ cells before the mobilization chemotherapy, samples of 16 patients were available. The mean proportion of c-kit expressing CD34+ cells in the bone marrow during G-CSF-stimulated reconstitution decreased 1.8-fold compared with baseline values. There was no difference in the proportion of BM- derived CD34+/Thy-1+ cells and CD34+/CD45RA+ cells between steady-state hematopoiesis and G-CSF-supported recovery. Our data suggest that during G-CSF-enhanced recovery, CD34+ cells in the PB are enriched with more primitive progenitor cells to evenly replenish the BM after the chemotherapy-related cytotoxic damage.     

Functional differences between CD38- and DR- subfractions of CD34+ bone marrow cells

Several studies have previously demonstrated enrichment in primitive progenitor cells in subfractions of CD34+ bone marrow (BM) cells not expressing CD38 or HLA-DR (DR) antigens. However, no studies have directly compared these two cell populations with regard to their content of primitive and more committed progenitor cells. Flow cytometric analysis of immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+CD38- and CD34+DR- progenitor subpopulations in that only 12% to 14% of total CD34+DR- and CD34+CD38- cells were double negative (CD34+CD38-DR-). Although the number of committed myeloid progenitor cells (colony-forming units granulocyte- macrophage) was reduced in both subpopulations, only CD34+CD38- cells were significantly depleted in committed erythroid progenitor cells (burst-forming units-erythroid). In single-cell assay, CD34+CD38- cells showed consistently poorer response to single as opposed to multiple hematopoietic growth factors as compared with unfractionated CD34+ cells, indicating that the CD34+CD38- subset is relatively enriched in primitive hematopoietic progenitor cells. Furthermore, CD34+CD38- and CD34+DR- cells, respectively, formed 3.2-fold and 1.6-fold more high proliferative potential colony-forming cell (HPP-CFC) colonies than did unfractionated CD34+ cells. Finally, CD34+CD38-DR- cells were depleted in HPP-CFCs as compared with CD34+CD38+DR+ cells. The results of the present study suggest that both the CD38- and DR- subfractions of CD34+ bone marrow cells are enriched in primitive hematopoietic progenitor cells, with the CD34+CD38- subpopulation being more highly enriched than CD34+DR- cells.     

Porcine brain microvascular endothelial cells support the in vitro expansion of human primitive hematopoietic bone marrow progenitor cells with a high replating potential: requirement for cell-to-cell interactions and colony-stimulating factors

Primary autologous as well as allogeneic and xenogeneic stroma will support human stem cell proliferation and differentiation for several months. In the present study, we investigated the capacity of porcine microvascular endothelial cells (PMVECs) together with combinations of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF] + stem factor [SCF], interleukin-3 [IL-3] + SCF + IL-6, and GM-CSF + IL-3 + SCF + IL-6) to support the expansion and development of purified human CD34+ bone marrow cells. In short-term cultures (7 days), the greatest expansion of nonadherent hematopoietic cells and clonogenic progenitors was seen with CD34+ cells in direct contact with PMVEC monolayers (PMVEC contact), followed by PMVEC noncontact and liquid suspension cultures, respectively. Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells (12.6-fold) occurred in PMVEC contact cultures treated with GM-CSF + IL-3 + SCF + IL-6, with similar increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), CFU-mix, erythroid burst-forming units (BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells. Moreover, the number of CD34+ CD38- and CD34+ CD38+ cells increased 148.1-fold and 8.0-fold, respectively. Replating studies show that cells from day 7 dispersed blast cell colonies generated on cytokine-treated PMVEC monolayers have a high replating potential for multilineage progenitor cells. In long- term PMVEC contact cultures, CD34+ cells seeded onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total calculated expansion of over 5,000,000-fold of nonadherent cells over 35 days in culture. Maximal clonogenic cell production was observed at day 28, with 6,353- fold for total CFC and comparable increases for CFU-GM, CFU-mix, CFU- blast, BFU-E, and CFU-Mk. The total number of CD34+ cells increased 2,584-fold at day 28. Furthermore, the extended growth kinetics of these cultures indicates that these phenotypically primitive progenitor cells are also functionally expanded on PMVEC monolayers. These results support the hypothesis that direct contact with a PMVEC monolayer supports the initial expansion of hematopoietic progenitor cells with a high replating potential and, possibly, a more primitive phenotype (CD34+, CD34+/CD38-).     

Expression of CD4 by human hematopoietic progenitors [see comments]

It has been recently reported that murine hematopoietic stem cells and progenitors express low levels of CD4. In this study, we have investigated by phenotypic and functional analysis whether the CD4 molecule was also present on human hematopoietic progenitors. Unfractionated marrow cells or immunomagnetic bead-purified CD34+ cells were analyzed by two-color fluorescence with an anti-CD4 and an anti- CD34 monoclonal antibody (MoAb). A large fraction (25% to 50%) of the CD34+ cells was weakly stained by anti-CD4 antibodies. Moreover, in further experiments analyzing the expression of CD4 in different subpopulations of CD34+ cells, we found that CD4 was predominantly expressed in phenotypically primitive cells (CD34+ CD38-/low CD71low Thy-1high, HLA-DR+/low). However, the presence of CD4 was not restricted to these primitive CD34+ cell subsets and was also detected in a smaller fraction of more mature CD34+ cells exhibiting differentiation markers. Among those, subsets with myelo-monocytic markers (CD13, CD33, CD14, and CD11b) have a higher CD4 expression than the erythroid or megakaryocytic subsets. In vitro functional analysis of the sorted CD34+ subsets in colony assays and long-term culture- initiating cell (LTC-IC) assays confirmed that clonogenic progenitors (colony-forming unit-granulocyte-macrophage, burst-forming unit- erythroid, and colony-forming unit-megakaryocyte) and LTC-IC were present in the CD4low population. However, most clonogenic progenitors were recovered in the CD4- subset, whereas the CD4low fraction was greatly enriched in LTC-IC. In addition, CD4low LTC-IC generated larger numbers of primitive clonogenic progenitors than did CD4- LTC-IC. These observations suggest that, in the progenitor compartment, the CD4 molecule is predominantly expressed on very early cells. The CD4 molecule present on CD34+ cells appeared identical to the T-cell molecule because it was recognized by three MoAbs recognizing different epitopes of the molecule. Furthermore, this CD4 molecule is also functional because the CD34+ CD4low cells are able to bind the human immunodeficiency virus (HIV) gp120. This observation might be relevant to the understanding of the mechanisms of HIV-induced cytopenias.     

Single leukapheresis products collected from healthy donors after the administration of granulocyte colony-stimulating factor contain ten-fold higher numbers of long-term reconstituting hematopoietic progenitor cells than conventional bone marrow allografts

Cytokine-mobilized peripheral blood progenitor cells (PBPCs) have been used successfully for hematopoietic reconstitution following allogeneic transplantation. The ease of harvest, the faster engraftment and the high yield of CD34+ cells have made this source of hematopoietic progenitor cells (HPCs) an attractive alternative to bone marrow (BM). In the present study we compared the engraftment potential of conventional BM allografts and single leukapheresis products (LPs) collected from healthy donors following the administration of granulocyte colony-stimulating factor (G-CSF). For this, lineage-committed and primitive HPCs were assessed by flow cytometry and by colony- and cobblestone area-forming cell (CFC, CAFC) assays. Mean numbers of CD34+ cells in LPs (n = 11) were similar to that of BM grafts (n = 12) (278+/-57 vs 227+/-34 x 10(6) CD34+ cells). The frequencies of CFCs, week 5 CAFCs and week 8 CAFCs were 1.6-, 8.4- and 10.3-fold higher in the CD34+ compartment of mobilized blood than that of marrow, resulting in significantly higher yields of clonogenic HPCs in LPs when compared to BM grafts. We conclude that G-CSF preferentially mobilizes clonogenic progenitors capable of short- and, in particular, longterm reconstitution, and that the engraftment potential of single LPs is superior to that of BM allografts. Hence, the use of PBPCs may be favorable for protocols that include graft manipulations with expected cell loss (eg T cell depletion, CD34+ selection). PBPCs may also be advantageous for gene therapy trials due to their high numbers of potential target cells (eg CAFCs).     

Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential

Embryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to 20% of CD34+ cells and isolate up to 10(7) CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell-derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase-positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (IL-7), and IL-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells.     

Changes in the growth properties of CD34+, CD38- bone marrow progenitors during human fetal development

We have previously described the isolation of separate populations of CD34+, CD38- stromal and hematopoietic progenitors cells within fetal bone marrow. The CD34+, CD38-, CD50+, HLA-DR+ population contained the majority of primitive hematopoietic progenitor cells, whereas stromal progenitors were contained within the CD34+, CD38-, CD50-, HLA-DR- population. In this study, we compared the frequencies and total numbers of clonogenic CD34+, CD38- stromal and hematopoietic cells as a function of fetal gestational age using single-cell fluorescent- activated cell sorting (FACS). At 14 weeks of gestation, 1/500 fetal bone marrow mononuclear cells were primitive hematopoietic CD34+, CD38- , HLA-DR+ progenitor cells, whereas 1/1,000 were stromal progenitors with the CD34+, CD38-, HLA-DR- phenotype. During fetal ontogeny there was a continuous, age-dependent decrease in the frequency of stromal progenitors, such that, at 24 weeks of gestation, only 1/100,000 of bone marrow cells had the CD34+, CD38-, HLA-DR- phenotype and were clonogenic stromal cells when isolated by FACS. In contrast, 1/250 bone marrow cells in a 24-week fetus had the CD34+, CD38-, HLA-DR+ phenotype and were clonogenic hematopoietic progenitors. The decrease in the frequency of stromal progenitors was a function of both a decreased frequency of cells with the CD34+, CD38-, HLA-DR- phenotype and a decrease in the growth potential of individual with this phenotype. The total numbers of mononuclear cells and the total numbers of hematopoietic progenitors in two fetal femurs increased in parallel, 100-fold, between 14 and 24 weeks of gestation. In contrast, the total numbers of clonogenic CD34+, CD38-, HLA-DR- stromal progenitor cells remained constant during this period. Although adult bone marrow samples contained stromal progenitor cells at a frequency of approximately 1/7,000 mononuclear cells, clonogenic stromal cells with the CD34+, CD38-, HLA-DR- phenotype could not be isolated by single- cell FACS from these samples. Thus, there are significant differences between the frequencies and biologic characteristics of stromal and hematopoietic stem cells during fetal and postnatal ontogeny.     

Persistence of human multilineage, self-renewing lymphohematopoietic stem cells in chimeric sheep

We have previously reported the ability of uncharacterized human bone marrow (BM) cells to engraft into preimmune fetal sheep, thereby creating sheep-human chimera suitable for in vivo examination of the properties of human hematopoietic stem cells (HSC). Adult human bone marrow CD34+ HLA-DR- cells have been extensively characterized in vitro and have been demonstrated to contain a number of primitive hematopoietic progenitor cells (PHPC). However, the capacity of such highly purified populations of human marrow CD34+ HLA-DR- cells to undergo in vivo self-renewal and multipotential lymphohematopoietic differentiation has not been previously demonstrated. To achieve that, human CD34+ HLA-DR- cells were transplanted in utero into immunoincompetent fetal sheep to investigate the BM-populating potential of these cells. Long-term chimerism, sustained human hematopoiesis, and expression of human cells belonging to all human blood cell lineages were demonstrated in two animals for more than 7 months' posttransplantation. Chimeric BM contained erythroid, granulocytic/monocytic, and megakaryocytic hematopoietic progenitor cells, as well as the primitive high proliferative potential colony- forming cell (HPP-CFC). Under a variety of in vitro experimental conditions, chimeric BM cells gave rise to human T cells expressing T- lymphocyte-specific markers, human natural killer (NK) cells, and human IgG-producing B cells. In vivo expansion and possibly self-renewal of transplanted PHPC was confirmed by the detection in chimeric BM 130 days' posttransplantation of CD34+ HLA-DR- cells, the phenotype of human cells constituting the stem-cell graft. These studies demonstrate not only the BM-populating capacity, multipotential differentiation, and most likely self-renewal capabilities of human CD34+ HLA-DR- cells, but also that this BM population contains human HSC. Furthermore, it appears that this animal model of xenogeneic stem-cell transplantation is extremely useful for in vivo examination of human hematopoiesis and the behavioral and functional characteristics of human HSC.     

Identification of a novel, human multilymphoid progenitor in cord blood

The earliest stages of lymphoid commitment from human pluripotent hematopoietic stem cells have not been defined. A clonogenic subpopulation of CD34(+)CD38(-) cord blood cells were identified that expressed high levels of the CD7 antigen and possessed only lymphoid potential. CD34(+)CD38(-)CD7(+) (CD7(+)) cells uniformly coexpressed CD45RA and HLA-DR; c-kit and Thy-1 expression was absent to low. Clonal analysis demonstrated that single CD7(+) cells could generate B cells, natural killer cells, and dendritic cells but were devoid of myeloid or erythroid potential. In contrast, control CD34(+)CD38(-)CD7(-) (CD7(-)) cells generated both lymphoid and myelo-erythroid cells. The lymphoid potential (generation of lymphoid progeny in bulk and single cell cultures) of CD7(+) cells was equivalent to that of the pluripotent CD7(-) cells. RNA expression studies showed that CD7(+) cells expressed PU.1 and GATA-3, but did not express Pax-5, terminal deoxynucleotide transferase, or CD3epsilon. In contrast to the previously described murine common lymphoid progenitor, the alpha chain of the receptor for interleukin-7 was not detected by fluorescence-activated cell sorting analysis or RNA polymerase chain reaction in CD7(+) cells. These studies identify a clonogenic lymphoid progenitor with both B-cell and natural killer cell lineage potential with a molecular profile that suggests a developmental stage more primitive than previously identified lymphoid progenitors. The CD7(+) phenotype distinguishes primitive human lymphoid progenitors from pluripotent stem cells, thus allowing the study of regulation of early human lymphopoiesis and providing an alternative to pluripotent stem cells for genetic manipulation and transplantation. (Blood. 2001;97:3683-3690)     

CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells.

CD109 is a monomeric cell surface glycoprotein of 170 kD that is expressed on endothelial cells, activated but not resting T-lymphocytes, activated but not resting platelets, leukemic megakaryoblasts, and a subpopulation of bone marrow CD34+ cells. Observing an apparent association between CD109 expression and the megakaryocyte lineage (MK), we sought to determine whether CD109 was expressed on MK progenitors. In fetal bone marrow (FBM), a rich source of MK progenitors, CD109 is expressed on a mean of 11% of CD34- cells. Fluorescence activated cell sorting (FACS) of FBM CD34+ cells into CD109+ and CD109- fractions revealed that the CD34+CD109+ subset contained virtually all assayable MK progenitors, including the colony-forming unit-MK (CFU-MK) and the more primitive burst-forming unit-MK (BFU-MK). The CD34+CD109+ subset also contained all the assayable burst-forming units-erythroid (BFU-E), 90% of the colony-forming units-granulocyte/macrophage (CFU-GM), and all of the more primitive mixed lineage colony-forming units (CFU-mix). In contrast, phenotypic analysis of the CD34+CD109- cells in FBM, adult bone marrow (ABM) and cytokine-mobilized peripheral blood (MPB) demonstrated that this subset comprises lymphoid-committed progenitors, predominantly of the B-cell lineage. CD109 was expressed on the brightest CD34 cells identifiable not only in FBM, but also in ABM and MPB indicating that the most primitive, candidate hematopoietic stem cells (HSC) might also be contained in the CD109+ subset. In long-term marrow cultures of FBM CD34+ cells, all assayable cobblestone area forming cell (CAFC) activity was contained within the CD109+ cell subset. Further phenotypic analysis of the CD34+CD109+ fraction in ABM indicated that this subset included candidate HSCs that stain poorly with CD38, but express Thy-1 (CD90) and AC133 antigens, and efflux the mitochondrial dye Rhodamine 123 (Rho123). When selected CD34+ cells were sorted for CD109 expression and Rho123 staining, virtually all CAFC activity was found in the CD109+ fraction that stained most poorly with Rho123. CD34+ cells were also sorted into Thy-1 CD109+ and Thy-1 CD109+ fractions and virtually all the CAFC activity was found in the Thy-1+CD109+ subset. In contrast, the Thy-1-CD109+ fraction contained most of the short-term colony-forming cell (CFC) activity. CD109, therefore, is an antigen expressed on a subset of CD34+ cells that includes pluripotent HSCs as well as all classes of MK and myelo-erythroid progenitors. In combination with Thy-1, CD109 can be used to identify and separate myelo-erythroid and all classes of MK progenitors from candidate HSCs.