Adult human bone marrow (ABM) is an important source of hematopoietic stem cells for transplantation in the treatment of malignant and nonmalignant diseases. However, in contrast to the recent progress that has been achieved with umbilical cord blood, methods to expand ABM stem cells for therapeutic applications have been disappointing. In this study, we describe a novel culture method that uses human brain endothelial cells (HUBECs) and that supports the quantitative expansion of the most primitive measurable cell within the adult bone marrow compartment, the nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cell (SRC). Coculture of human ABM CD34+ cells with brain endothelial cells for 7 days supported a 5.4-fold increase in CD34+ cells, induced more than 95% of the CD34+CD38 subset to enter cell division, and produced progeny that engrafted NOD/SCID mice at significantly higher rates than fresh ABM CD34+ cells. Using a limiting dilution analysis, we found the frequency of SRCs within fresh ABM CD34+ cells to be 1 in 9.9 × 105 cells. Following HUBEC culture, the estimated frequency of SRCs increased to 1 in 2.4 × 105cells. All mice that received transplants of HUBEC-cultured cells showed B-lymphoid and myeloid differentiation, indicating that a primitive hematopoietic cell was preserved during culture. Noncontact HUBEC cultures also maintained SRCs at a level comparable to contact HUBEC cultures, suggesting that cell-to-cell contact was not required. These data demonstrate that human brain endothelial cells possess a unique hematopoietic activity that increases the repopulating capacity of adult human bone marrow.

The development of ex vivo culture methods that promote the expansion of adult human bone marrow (ABM) stem cells would have direct application in clinical gene therapy and stem cell transplantation. However, results obtained from stroma-based1 and stroma-free ex vivo culture systems2-5 have been disappointing, owing to insufficient activation of primitive CD34+CD38 cells, cell differentiation, and a loss of repopulating capacity following short-term culture.6 Moreover, increased CD34+ cell numbers, colony-forming cells (CFCs), and long-term culture initiating cells (LTC-ICs) are not quantitative indicators of in vivo repopulating potential.7-10Therefore, the importance of evaluating ex vivo cultured cells in an in vivo repopulation model has been emphasized.8 

The nonobese diabetic/severe combined immunodeficient (NOD/SCID) model system has been used to measure the long-term reconstitution potential of ex vivo–expanded human lymphohematopoietic stem cells.7-10 SCID-repopulating cells (SRCs) are enriched in human cord blood (CB) as compared with adult ABM and mobilized peripheral blood11,12 and are most highly concentrated within the CD34+CD38population.8 SRCs are considered to be biologically more primitive than assayable LTC-IC and CFC progenitors,1,9,10,13 which are found in both the CD34+CD38+ and the CD34+CD38 pools.8 As further evidence, gene transduction studies have shown that LTC-ICs and CFCs are readily transduced but contribute little to NOD/SCID engraftment.2 7 

When human hematopoietic stem cells (HSCs) are cocultured in contact with bone marrow stroma or conditioned medium from stromal cultures, a percentage of LTC-ICs and CFCs can be expanded in vitro over several weeks.14-17 Similarly, bone marrow–, umbilical vein–, and yolk sac–derived endothelial cell cultures elaborate growth factors that regulate hematopoiesis18-20 and support the proliferation of myeloid, erythroid, and megakaryocytic progenitors.19,20 We have previously demonstrated that a porcine brain microvascular endothelial cell line (PMVEC) plus cytokines was capable of supporting a robust expansion of human CD34+CD38 progenitors21,22 while maintaining cells capable of repopulating SCID human (SCID-Hu) bone23 as well as cells capable of rescuing lethally irradiated baboons.24 In these studies, we did not quantify the frequency of repopulating cells in the transplanted grafts.

Recent studies using rigorous limiting dilution analyses have demonstrated that both stroma-containing and stroma-free culture conditions can support the quantitative expansion of SRCs within human CB.25-28 However, the ex vivo expansion of human ABM stem cells under similarly stringent conditions has not been demonstrated. In fact, a recent limiting dilution analysis demonstrated a 6-fold decline in SRCs within human ABM during short-term culture with ABM stroma.1 In this study, using quantitative limiting dilution analysis, we demonstrate that the number of engraftable SRCs within human ABM increases following coculture with primary human brain endothelial cells (HUBECs). The HUBEC ex vivo culture system has potential application in the expansion of ABM stem cells for clinical transplantation and will also be a new resource for the identification of molecules that affect stem-cell self-renewal.

Isolation of primary HUBECs

Vessel segments (smaller than 10 cm) from the central nervous system (CNS) and outside the CNS (renal artery) were collected from cadavers within 12 hours after death under an approved tissue-procurement protocol. Vessel segments were placed in complete endothelial cell culture medium containing M199 (GIBCO/BRL, Gaithersburg, MD), 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT), 100 μg/mLl-glutamine, 50 μg/mL heparin, 30 μg/mL endothelial cell growth supplement (Sigma, St Louis, MO), 100 U/mL penicillin, and 100 μg/mL streptomycin.

Vessels were incised longitudinally and oriented in such a fashion that the lumen side contacted the dish surface during in vitro culture. Well-developed endothelial cell colonies were evident by day 14, and confluent monolayers were achieved by day 30 of culture. Colonies were fed weekly with complete medium, and several passages of the primary cells were banked.

CD34+ cells plus HUBEC coculture

Purified human ABM CD34+ cells were obtained from Poietics Technologies (Gaithersburg, MD). HUBECs were subcultured at 1 × 105 cells per well in gelatin-coated 6-well plates (Costar, Cambridge, MA) as previously described.22After 72 hours, HUBEC monolayers were washed with phosphate-buffered saline (PBS), and the spent medium was replaced with ex vivo expansion culture medium (5 mL per well) consisting of Iscove modified Dulbecco medium (IMDM) (GIBCO/BRL) containing 10% FBS, 200 μMl-glutamine, 2 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 5 ng/mL interleukin 3 (IL-3), 5 ng/mL IL-6, 120 ng/mL stem cell factor (SCF), and 50 ng/mL flt-3 ligand (R & D Systems, Minneapolis, MN) to each well. Purified ABM CD34+ cells (1 × 105) were added to each well, and cultures were maintained at 37°C in 5% CO2 atmosphere. After 7 days of culture, nonadherent cells were harvested by washing the monolayers gently with warm complete culture medium. In the HUBEC noncontact cultures, ABM CD34+ cells were plated in the upper compartment of the culture well, separated from HUBEC monolayers by transwell inserts (0.4 μm) (Costar).

Immunofluorescence staining and cell cycle analysis

ABM CD34+ cells and cultured cells were stained with monoclonal antibodies against CD34–fluorescein isothiocyanate (FITC) and CD38–phycoerythrin (PE) (Becton Dickinson [BD], San Jose, CA) and analyzed by Epics Elite fluorescence-activated cell sorter (FACS) (Coulter, Hialeah, FL). Controls consisted of isotype-matched monoclonal antibodies (mAbs). We performed the surface, intracellular, DNA (SID) cell cycle analysis as previously described29 using anti-CD34–allophycocyanin (APC) (BD), CD38-PE (BD), Ki-67–FITC (Immunotech, Westbrook, ME), and 7-amino-actinomycin D (7-AAD) (Sigma). Isotype controls were performed in parallel for each sample.

In vitro methylcellulose colony forming assays

Purified ABM CD34+ cells and ex vivo cultured cells (5 to 500 × 102) were cultured in 35-mm culture dishes (Miles Laboratories, Naperville, IL) as previously described.22 Culture media consisted of 1 mL IMDM, 1% methylcellulose, 30% FBS, 10 U/mL erythropoietin, 2 ng/mL GM-CSF, 10 ng/mL IL-3, and 120 ng/mL SCF. At day 14, we evaluated triplicate cultures to determine the number of colonies (larger than 50 cells) per dish. NOD/SCID marrow cells were washed × 2 and placed (1 × 105) in methylcellulose containing culture media containing the above-noted human cytokines and analyzed at day 14 for evidence of human colonies.

Transplantation of fresh ABM CD34+ cells and HUBEC-cultured cells in NOD/SCID mice

NOD/SCID mice30 received transplants of either fresh purified ABM CD34+ cells or the progeny of ABM CD34+ cells cultured with HUBECs supplemented with GM-CSF plus IL-3 plus IL-6 plus SCF plus flt-3 ligand over a range of doses designed to achieve no engraftment in a significant fraction. To avoid donor variability, HUBEC cultures were established with the identical ABM CD34+ cells as used for transplantation into mice designated the “fresh ABM CD34+” group. Cells were transplanted via tail vein injection after irradiating the mice with 300 cGy by means of a 137Cs source as previously described.31 The mice received no CD34accessory cells or exogenous cytokines to facilitate engraftment. Mice were killed at week 8, and marrow samples were obtained by flushing their femurs and tibias with IMDM at 4°C.

Flow cytometric analysis was performed as previously described with the use of commercially available monoclonal antibodies against human leukocyte differentiation antigens to identify engrafted human leukocytes and discriminate their hematopoietic lineages.31 Immunofluoresence staining of marrow cells was performed following our previously published procedures.31 

Statistical analysis

For purposes of our limiting dilution assays, we scored a mouse that underwent transplantation as positive if at least 1% of the marrow cells expressed human CD45 via FACS analysis, based upon the engraftment criteria established by Ueda et al.27 We calculated the SRC frequency in each cell source using the maximum likelihood estimator as described previously by Taswell32 for the single-hit Poisson model.12,27 The χ2 provides a measure of the legitimacy of using pooled data and of the validity of applying the single-hit model.12 30 We calculated confidence intervals for the frequencies using the profile likelihood method. As a confirmation of the maximum likelihood estimator, we also applied a minimum χ2 estimator to the pooled data.

HUBEC coculture supports ABM progenitor cell proliferation and expansion

HUBECs displayed cobblestone morphology at confluence and more than 90% expressed von Willebrand factor, but we did not detect CD34 or CD38 expression by flow cytometry (data not shown). The effects of HUBEC contact and noncontact coculture, liquid suspension culture, and human nonbrain endothelial cell culture on CD34+ cell expansion and CFC generation were compared. All cultures were supplemented with GM-CSF plus IL-3 plus IL-6 plus SCF plus flt-3 ligand because our previous studies indicated that this combination optimized the expansion of ABM CD34+ cells.21 22HUBEC culture supported a 16.1-fold increase in total cells, a 5.4-fold increase in CD34+ cells, and a 212-fold increase in the CD34+CD38 subset (n = 8) (Table1). CD34+CD38cells increased from 1.6% of the total population at day 0 to 21.6% at day 7 and constituted 64% of the day-7 CD34+ cell pool. HUBEC noncontact cultures supported a 16.4-fold expansion of total cells, a 2.8-fold increase in CD34+ cells, and a 35-fold increase in the CD34+CD38 population. In contrast, liquid suspension cultures and nonbrain endothelial cell cultures supported similar increases in total cells and CD34+ cells, but neither maintained CD34+CD38 cells at day 7 (Figure 1A-E) (Table 1).

Table 1.

HUBEC coculture promotes the expansion of human CD34+ subsets and CFCs compared with controls

ConditionsCell yieldNo. cells procured × 105No. CFCs × 104Total
CD34+CD34+CD38+(%)CD34+CD38(%)CFU-GMsBFU-EsCFU-Mix's
Input 5.0 5.0 4.91 ± 0.07 (98.4) 0.08 ± 0.04 (1.6) 3.7 ± 0.9 0.6 ± 0.3 0.5 ± 0.2 4.7 ± 1.4 
HUBECs 81.0 ± 3.4 (16.1-fold) 27.0 ± 1.1 (5.4-fold) 9.0 ± 0.4 (12.3) 17.0 ± 0.5 (21.6) 56.0 ± 2.2 4.8 ± 0.6 2.6 ± 0.3 63.4 ± 2.5 
HUBECs, noncontact 82.0 ± 3.7 (16.4-fold) 14.0 ± 0.1 (2.8-fold) 10.4 ± 0.3 (13.0) 2.8 ± 0.1 (3.4) 30.0 ± 1.3 6.0 ± 0.5 4.3 ± 0.9 40.3 ± 0.5 
Stroma-free 51.0 ± 1.3 (10.2-fold) 15.1 ± 0.7 (3.0-fold) 15.1 ± 0.6 (29.6) 0.02 ± 0.02 (0.1) 15.8 ± 4.5 0.7 ± 0.7 0.5 ± 0.9 17.1 ± 4.6 
Nonbrain ECs 52.0 ± 2.8 (10.4-fold) 18.1 ± 12.6 (3.6-fold) 18.1 ± 12.6 (34.8) 0 (0) 22.2 ± 4.9 4.8 ± 0.3 0.6 ± 0.3 27.7 ± 6.2 
ConditionsCell yieldNo. cells procured × 105No. CFCs × 104Total
CD34+CD34+CD38+(%)CD34+CD38(%)CFU-GMsBFU-EsCFU-Mix's
Input 5.0 5.0 4.91 ± 0.07 (98.4) 0.08 ± 0.04 (1.6) 3.7 ± 0.9 0.6 ± 0.3 0.5 ± 0.2 4.7 ± 1.4 
HUBECs 81.0 ± 3.4 (16.1-fold) 27.0 ± 1.1 (5.4-fold) 9.0 ± 0.4 (12.3) 17.0 ± 0.5 (21.6) 56.0 ± 2.2 4.8 ± 0.6 2.6 ± 0.3 63.4 ± 2.5 
HUBECs, noncontact 82.0 ± 3.7 (16.4-fold) 14.0 ± 0.1 (2.8-fold) 10.4 ± 0.3 (13.0) 2.8 ± 0.1 (3.4) 30.0 ± 1.3 6.0 ± 0.5 4.3 ± 0.9 40.3 ± 0.5 
Stroma-free 51.0 ± 1.3 (10.2-fold) 15.1 ± 0.7 (3.0-fold) 15.1 ± 0.6 (29.6) 0.02 ± 0.02 (0.1) 15.8 ± 4.5 0.7 ± 0.7 0.5 ± 0.9 17.1 ± 4.6 
Nonbrain ECs 52.0 ± 2.8 (10.4-fold) 18.1 ± 12.6 (3.6-fold) 18.1 ± 12.6 (34.8) 0 (0) 22.2 ± 4.9 4.8 ± 0.3 0.6 ± 0.3 27.7 ± 6.2 

Purified human ABM CD34+ cells (5 × 105) were plated per culture treatment (n = 8) as described in “Materials and methods.” Nonadherent cells were harvested on day 7 of culture and analyzed via flow cytometry. Numbers in parentheses in the cell-yield and CD34+ columns reflect the fold-increase in these populations following culture. Numbers in parentheses in the CD34+ CD38+ and CD34+CD38 columns indicate the percentage of each population as a subset of the total population. CFC values represent the total number of CFC per culture.

CFU-GMs indicates GM colony-forming units; BFU-Es, erythroid burst-forming units; CFU-Mix's, mixed CFUs.

HUBEC coculture supported a 15.1-fold increase in GM colony-forming units (CFU-GMs), a 5.2-fold increase in mixed CFUs (CFU-Mix's), and an 8.0-fold increase in erythroid burst-forming units (BFU-Es) compared with input values, and this CFC activity was significantly greater than that produced by liquid suspension cultures and nonbrain endothelial cell cultures (Table 1). Noncontact HUBEC cultures supported an expansion of total CFCs that was greater than either liquid suspension or nonbrain endothelial cell culture, but optimal CFC production occurred in the HUBEC contact cultures.

HUBEC contact and noncontact cultures induced a high percentage of quiescent ABM CD34+CD38 cells to enter cell division by day 7. At day 0, 95.7% ± 3.9% of the CD34+CD38 population resided in G0; 3.9% ± 3.2% in G1; and 0.7% ± 0.7% in G2/S/M phase. After 7 days of HUBEC coculture, 62.3% ± 9.9% of the CD34+CD38cells had entered G1; 33.3% ± 7.6% were in G2/S/M phase; and only 3.9% ± 2.7% remained in G0 (Figure 2). Similarly, noncontact HUBEC cultures induced 49.1% ± 2.4% cells into G1, 25.3% ± 6.2% into G2/S/M phase, and only 25.1% ± 3.8% into G0. In contrast, we could not perform cell cycle analysis of CD34+CD38 cells from liquid suspension and nonbrain endothelial cell cultures owing to the undetectable frequency of CD34+CD38 cells at day 7.

Fig. 1.

Phenotypic analysis of bone marrow CD34+cells following ex vivo culture.

Purified human CD34+ cells were plated on confluent HUBEC monolayers, stroma-free liquid suspension cultures, nonbrain endothelial cell monolayers, and noncontact HUBEC cultures in the presence of optimal concentrations of GM-CSF plus IL-3 plus IL-6 plus SCF plus flt-3 ligand for 7 days. (A) The phenotype of untreated purified bone marrow CD34+ cells at day 0. (B) HUBEC-cultured bone marrow cells at day 7, demonstrating a high percentage of CD34+ and CD34+CD38cells. (C-D) Stroma-free liquid suspension cultures (C) and nonbrain endothelial cell cocultures, demonstrating loss of CD34+CD38 phenotype cells (D). (E) HUBEC noncontact cultures, showing preservation of cells with CD34+CD38 phenotype. Harvested nonadherent cells were stained with FITC-conjugated CD34 mAb and PE-conjugated CD38 mAb and analyzed by FACS. Log fluorescence distribution of CD34 expression is shown along the x-axis, and CD38 expression along the y-axis. Cursor lines indicate the nonspecific staining levels of isotype-matched control mAbs.

Fig. 1.

Phenotypic analysis of bone marrow CD34+cells following ex vivo culture.

Purified human CD34+ cells were plated on confluent HUBEC monolayers, stroma-free liquid suspension cultures, nonbrain endothelial cell monolayers, and noncontact HUBEC cultures in the presence of optimal concentrations of GM-CSF plus IL-3 plus IL-6 plus SCF plus flt-3 ligand for 7 days. (A) The phenotype of untreated purified bone marrow CD34+ cells at day 0. (B) HUBEC-cultured bone marrow cells at day 7, demonstrating a high percentage of CD34+ and CD34+CD38cells. (C-D) Stroma-free liquid suspension cultures (C) and nonbrain endothelial cell cocultures, demonstrating loss of CD34+CD38 phenotype cells (D). (E) HUBEC noncontact cultures, showing preservation of cells with CD34+CD38 phenotype. Harvested nonadherent cells were stained with FITC-conjugated CD34 mAb and PE-conjugated CD38 mAb and analyzed by FACS. Log fluorescence distribution of CD34 expression is shown along the x-axis, and CD38 expression along the y-axis. Cursor lines indicate the nonspecific staining levels of isotype-matched control mAbs.

Close modal
Fig. 2.

HUBEC culture induces cell division within quiescent ABM CD34+CD38 cells.

(A) The dot plot shows the 7-AAD versus Ki-67 FITC profile for steady-state bone marrow CD34+CD38 cells, with the majority of the population residing in G0. (B) CD34+CD38 cells were FACS-sorted from 7-day HUBEC cocultures and stained for 7-AAD versus Ki-67 FITC expression, demonstrating that the majority of the CD34+CD38 population had entered cell cycle. Cell cycle analysis of CD34+ cells was performed by means of the SID method described in “Materials and methods.”

Fig. 2.

HUBEC culture induces cell division within quiescent ABM CD34+CD38 cells.

(A) The dot plot shows the 7-AAD versus Ki-67 FITC profile for steady-state bone marrow CD34+CD38 cells, with the majority of the population residing in G0. (B) CD34+CD38 cells were FACS-sorted from 7-day HUBEC cocultures and stained for 7-AAD versus Ki-67 FITC expression, demonstrating that the majority of the CD34+CD38 population had entered cell cycle. Cell cycle analysis of CD34+ cells was performed by means of the SID method described in “Materials and methods.”

Close modal

HUBEC-cultured cells engraft NOD/SCID mice at a higher frequency than fresh ABM CD34+ cells

NOD/SCID mice received transplants of either fresh ABM CD34+ cells (n = 47) or the progeny of ABM CD34+ cells cultured with HUBEC × 7 days (n = 47) over a range of doses, which resulted in nonengraftment in a fraction of the mice.

Transplantation of 1 × 105 fresh ABM CD34+cells resulted in no engraftment in 10 mice, whereas transplantation of 5 × 105 to 1 × 106 ABM CD34+cells resulted in engraftment in only 10 of 22 recipients (45%) at low human CD45+ (huCD45+) cell levels (mean, 3.3%) (Figure 3A-C). At a dose of 1.5 × 106 ABM CD34+ cells, 7 of 7 mice that underwent transplantation showed human cell engraftment, suggesting that nonlimiting numbers of SRCs were present at that dose (Figure 3D). When the progeny of HUBEC cultures over the same dose range were transplanted, the rate of NOD/SCID engraftment increased (Figure 3A-C). The progeny of 1 × 105 ABM CD34+ cells cultured with HUBECs engrafted in 2 of 10 mice, and the progeny of 5 × 105 to 1 × 106 ABM CD34+cells engrafted in 21 of 22 mice (96%) at high levels (mean, 11.7% huCD45+ cells) (Figure 3A-C). Twelve mice receiving transplants of 5 × 105 to 1 × 106 fresh ABM CD34+ cells showed no human cell engraftment, but all 12 mice receiving transplants of the HUBEC-cultured progeny of these ABM CD34+ cells demonstrated engraftment of at least 1% huCD45+ cells. At a dose of 1.5 × 106ABM CD34+ cells, HUBEC-cultured progeny engrafted in 7 of 7 mice at levels of huCD45+ cell engraftment equivalent to fresh ABM CD34+ cells (Figure 3D).

Fig. 3.

Human cell engraftment in NOD/SCID mice receiving transplants of limiting doses of ABM CD34+ cells and HUBEC-cultured progeny.

(A) First, 1 × 105 ABM CD34+ cells (left) or their progeny following HUBEC culture (right) were transplanted into NOD/SCID mice. The level of human cells present in the murine bone marrow at 8 weeks was then determined by flow cytometric analysis of human CD45 expression. Panel B shows 5 × 105 ABM CD34+ cells or their HUBEC-cultured progeny; panel C, 1 × 106 ABM CD34+ cells versus HUBEC-cultured progeny; and panel D, 1.5 × 106 ABM CD34+ cells versus HUBEC-cultured progeny. Panel E shows the engraftment of progeny of 1.5 × 106 ABM CD34+ cells cultured with HUBEC noncontact cultures as well as the engraftment of the progeny of 3 × 106 ABM CD34+ cells cultured with GM-CSF plus IL-3 plus IL-6 plus SCF plus flt-3 ligand in the absence of HUBECs.

Fig. 3.

Human cell engraftment in NOD/SCID mice receiving transplants of limiting doses of ABM CD34+ cells and HUBEC-cultured progeny.

(A) First, 1 × 105 ABM CD34+ cells (left) or their progeny following HUBEC culture (right) were transplanted into NOD/SCID mice. The level of human cells present in the murine bone marrow at 8 weeks was then determined by flow cytometric analysis of human CD45 expression. Panel B shows 5 × 105 ABM CD34+ cells or their HUBEC-cultured progeny; panel C, 1 × 106 ABM CD34+ cells versus HUBEC-cultured progeny; and panel D, 1.5 × 106 ABM CD34+ cells versus HUBEC-cultured progeny. Panel E shows the engraftment of progeny of 1.5 × 106 ABM CD34+ cells cultured with HUBEC noncontact cultures as well as the engraftment of the progeny of 3 × 106 ABM CD34+ cells cultured with GM-CSF plus IL-3 plus IL-6 plus SCF plus flt-3 ligand in the absence of HUBECs.

Close modal

To assess the capacity of noncontact HUBEC cultures to maintain SRCs, we also transplanted into NOD/SCID mice the progeny of ABM CD34+ cells that were cultured in this manner. The progeny of 1.5 × 106 ABM CD34+ cells plated in HUBEC noncontact cultures engrafted in 7 of 7 mice at high levels (mean, 62.8% huCD45+) comparable to the engraftment observed with the progeny of contact HUBEC-cultured cells transplanted at the same dose (Figure 3E). As a control, we also transplanted into mice the progeny of ABM CD34+ cells that were plated in liquid suspension cultures supplemented with the identical cytokines for 7 days. None of the 3 mice in this group showed human cell engraftment or human CFC activity (Figure 3E).

HUBEC-cultured cells engraft in NOD/SCID mice with multilineage differentiation

Figure 4A shows human CD45+ cell engraftment within a representative mouse that received a transplant of fresh ABM CD34+ cells (1 × 106) versus an animal receiving a transplant of the progeny of the same dose of ABM CD34+ cells following HUBEC culture. Detailed FACS analysis demonstrated lymphoid and myeloid differentiation in mice receiving transplants of limiting doses of HUBEC-cultured cells (Figure 4B). The proportion of CD34+ cells, CD19+ B cells, and CD13+ myeloid cells was highly similar within mice receiving transplants of limiting doses of HUBEC-cultured cells as compared with mice engrafted by means of fresh ABM CD34+cells, indicating that a highly primitive repopulating cell was sustained during HUBEC culture (Table2). Detection of human CFCs within NOD/SCID mice correlated closely with the huCD45+ cell engraftment that we observed. Mice receiving transplants of the progeny of 1 × 106 ABM CD34+ cells cultured with HUBECs demonstrated multilineage human CFC activity that was 41-fold greater than the human CFC activity within mice receiving transplants of the same dose of fresh ABM CD34+ cells (Table 2).

Fig. 4.

Phenotypic analysis of HUBEC-cultured cells engrafted in the bone marrow of NOD/SCID mice.

(A) Expression of human CD45+ cells within the bone marrow of a control NOD/SCID mouse that did not receive a transplant (top); expression of human CD45+ cells within the bone marrow of a NOD/SCID mouse that received a transplant of 1 × 106fresh ABM CD34+ cells (middle); and expression of human CD45+ cells within the bone marrow of a NOD/SCID mouse that received a transplant of the progeny of 1 × 106 ABM CD34+ following coculture with HUBECs (bottom). Isotype controls are shown at left. (B) Lineage distribution of engrafted human cells within a representative mouse that received a transplant of HUBEC-cultured cells. Panel Bi, murine marrow stained with isotype control IgG1-FITC and IgG1–peridinin chlorophyll A protein (IgG1-PerCP). Panel Bii, staining of murine marrow with anti-human CD45-PerCP and anti-murine CD45-FITC, showing both human and murine populations. Panel Biii, isotype staining with IgG1-FITC and IgG1-PE. Panel Biv, expression of human CD34-PE and CD38-FITC on engrafted cells within murine marrow. Panel Bv, staining with anti-CD19 and anti-CD3, demonstrating CD19 expression on engrafted human cells. Panel Bvi, expression of human CD33 and human CD13 on engrafted cells within the marrow.

Fig. 4.

Phenotypic analysis of HUBEC-cultured cells engrafted in the bone marrow of NOD/SCID mice.

(A) Expression of human CD45+ cells within the bone marrow of a control NOD/SCID mouse that did not receive a transplant (top); expression of human CD45+ cells within the bone marrow of a NOD/SCID mouse that received a transplant of 1 × 106fresh ABM CD34+ cells (middle); and expression of human CD45+ cells within the bone marrow of a NOD/SCID mouse that received a transplant of the progeny of 1 × 106 ABM CD34+ following coculture with HUBECs (bottom). Isotype controls are shown at left. (B) Lineage distribution of engrafted human cells within a representative mouse that received a transplant of HUBEC-cultured cells. Panel Bi, murine marrow stained with isotype control IgG1-FITC and IgG1–peridinin chlorophyll A protein (IgG1-PerCP). Panel Bii, staining of murine marrow with anti-human CD45-PerCP and anti-murine CD45-FITC, showing both human and murine populations. Panel Biii, isotype staining with IgG1-FITC and IgG1-PE. Panel Biv, expression of human CD34-PE and CD38-FITC on engrafted cells within murine marrow. Panel Bv, staining with anti-CD19 and anti-CD3, demonstrating CD19 expression on engrafted human cells. Panel Bvi, expression of human CD33 and human CD13 on engrafted cells within the marrow.

Close modal
Table 2.

HUBEC-cultured cells generate multilineage human cell differentiation and human progenitor cell colonies within the marrow of NOD/SCID mice that underwent transplantation

Culture
conditions
Human CFCs formed from ABM of NOD/SCID micePercentage of engrafted CD45+ cells expressing specified CD cells
GMEMixTotalCD34CD19CD13
Fresh ABM        
CD34+cells 0.8 ± 0.5 0.2 ± 0.2 0.2 ± 0.2 1.2 ± 0.8 28.2 ± 3.6 89.9 ± 4.1 9.5 ± 3.4 
HUBECs 44.2 ± 15.9 3.4 ± 1.7 1.6 ± 1.5 49.0 ± 18.5 32.3 ± 9.4 77.7 ± 16.7 19.3 ± 7.7 
Culture
conditions
Human CFCs formed from ABM of NOD/SCID micePercentage of engrafted CD45+ cells expressing specified CD cells
GMEMixTotalCD34CD19CD13
Fresh ABM        
CD34+cells 0.8 ± 0.5 0.2 ± 0.2 0.2 ± 0.2 1.2 ± 0.8 28.2 ± 3.6 89.9 ± 4.1 9.5 ± 3.4 
HUBECs 44.2 ± 15.9 3.4 ± 1.7 1.6 ± 1.5 49.0 ± 18.5 32.3 ± 9.4 77.7 ± 16.7 19.3 ± 7.7 

NOD/SCID mice received transplants of either 1 × 106 ABM CD34+ cells or the progeny of this dose of ABM CD34+ cells following 7 days of HUBEC culture as described in “Materials and methods.” The CFC content in each group was determined from triplicate cultures of 1 × 105 NOD/SCID marrow cells per dish under procedures described in “Materials and methods” (n = 11). The numbers shown under the CD34, CD19, and CD13 columns indicate the percentage of engrafted human CD45+ cells that expressed the particular differentiation antigen. For animals receiving transplants of fresh ABM CD34+ cells, we were able to analyze only those mice with human cell engraftment ≥ 1%.

HUBEC coculture increases the frequency of SRCs within human bone marrow

For statistical analysis, we pooled data from the limiting dilution assays of fresh ABM CD34+ cells and HUBEC-cultured cells, according to methods previously described.12,27 We calculated the frequency of SRCs using the maximum likelihood estimator.32 The value of χ2 in all cases was not statistically significant (P > .10), demonstrating internal consistency in our assays and allowing pooling of the data. The frequency of SRCs within fresh ABM CD34+ cells was 1 in 9.9 × 105cells (95% confidence interval [CI], 1/650 000 − 1/1 600 000) (Figure 5A). The SRC frequency within HUBEC-cultured cells was significantly higher, at 1 in 240 000 cells (CI, 1/140 000 − 1/410 000) (Figure 5B). Therefore, coculture of adult human ABM CD34+ cells with HUBEC monolayers supported a 4.1-fold increase in SRC. As further confirmation of the validity of applying the single-hit Poisson model to our limiting dilution assay, we also estimated the frequency of SRCs using the minimum χ2 estimation.12 Again, χ2 was not significant in all cases (P > .20).

Fig. 5.

HUBEC-culture increases the frequency of SRCs within adult human bone marrow

. (A) NOD/SCID mice (n = 47) received transplants of fresh ABM CD34+ cells over a range of doses, and the engraftment frequencies at each dose are plotted. The resultant curve indicates the estimated frequency of SRCs within this population. (B) NOD/SCID mice (n = 47) received transplants of the progeny of ABM CD34+cells cultured with HUBECs plus GM-CSF plus IL-3 plus IL-6 plus SCF plus flt-3 ligand. The engraftment frequencies are plotted at each dose, and the resultant curve indicates the frequency of SRCs within this population. The numbers shown within each box indicate the calculated frequency of SRC using the maximum likelihood estimator.

Fig. 5.

HUBEC-culture increases the frequency of SRCs within adult human bone marrow

. (A) NOD/SCID mice (n = 47) received transplants of fresh ABM CD34+ cells over a range of doses, and the engraftment frequencies at each dose are plotted. The resultant curve indicates the estimated frequency of SRCs within this population. (B) NOD/SCID mice (n = 47) received transplants of the progeny of ABM CD34+cells cultured with HUBECs plus GM-CSF plus IL-3 plus IL-6 plus SCF plus flt-3 ligand. The engraftment frequencies are plotted at each dose, and the resultant curve indicates the frequency of SRCs within this population. The numbers shown within each box indicate the calculated frequency of SRC using the maximum likelihood estimator.

Close modal

Bone marrow transplantation is a curative therapy for an increasing number of malignant and nonmalignant diseases.33 However, the ex vivo expansion of adult bone marrow for application in gene therapy, immune tolerance induction,34 and other purposes has been unsuccessful owing to the differentiation and cell death that occur when these cells are exposed to cytokines.1-5 In this study, using a limiting dilution analysis, we have demonstrated for the first time that the SCID-repopulating cell numbers within adult ABM can be quantitatively increased via ex vivo coculture with primary HUBECs. Coculture of ABM CD34+ cells with HUBECs supplemented with GM-CSF plus IL-3 plus IL-6 plus SCF plus flt-3 ligand induced greater than 95% of the CD34+CD38 population to enter cell division and supported a 4.1-fold increase in SRCs as compared with starting ABM CD34+ populations. Since stem cell division and maintenance of stem cell repopulating capacity are requirements for successful retroviral gene transfer, the HUBEC culture method may provide significant advantages for clinical gene therapy protocols. Although other factors, such as the expression of retroviral receptors on target stem cells,35 are also important predictors of the success of retroviral gene therapy, our previous investigations have shown that coculture of human ABM CD34+ cells with a PMVEC increased the gene transfer efficiency into the CD34+CD38 subset in the absence of measurable increases in the expression of retroviral receptors.36 We are currently examining the effect of HUBEC coculture on the gene transfer efficiency into BM CD34+CD38 cells and long-term repopulating cells in the NOD/SCID model.

In contrast to the results presented here, previous studies have indicated that the ex vivo culture of ABM stem cells results in a decline in repopulating capacity.1,37 Gan et al1 reported that 1-week culture of human ABM mononuclear cells (MNCs) with ABM stroma caused a 6-fold decline in SRCs as compared with unmanipulated ABM MNCs. Studies of mouse ABM cultures have demonstrated similar losses in the recovery of long-term repopulating cells after 3 to 4 weeks in culture.37 Of note, both the murine studies and the studies by Gan et al were performed in the absence of exogenous cytokines, which would have been expected to drive differentiation.4-6 In our studies, we supplemented HUBEC monolayers with a cytokine combination, which maximally induced progenitor cell division, and despite this, we observed a measurable increase in SRCs over time. Since exposure to GM-CSF plus IL-3 plus IL-6 plus SCF plus flt-3 ligand in the absence of HUBECs caused a decline in detectable SRCs over 7 days of culture, we postulate that HUBECs may have protected ABM SRCs from differentiation during exposure to cytokines while also supporting the self-renewal of this primitive population.

Although steady-state ABM CD34+CD38 cells are highly enriched for SRCs,8 we observed no correlation between the observed increase in SRCs (4.1-fold) and the larger increase in CD34+CD38 cells during culture (212-fold). This may be explained, in part, by a down-modulation of CD38 expression on committed progenitors that may have occurred during culture. Dorrell et al38 demonstrated that a significant percentage of cord blood CD34+CD38+ cells acquired a CD34+CD38 phenotype during 4-day culture with fibronectin plus IL-6 plus SCF plus G-CSF plus flt-3 ligand, and a depletion of retinoids within the culture may account for this result.39 In addition, apoptotic events associated with cytokine deprivation of transplanted CD34+CD38 cells40 may contribute to their loss of repopulating capacity. Similarly, Glimm et al41 and others3,42 43 have demonstrated that cell cycle–associated defects may adversely affect the ability of subpopulations of proliferating CD34+CD38cells to contribute to in vivo engraftment. Our preliminary investigations indicate that coculture of sorted ABM CD34+CD38 cells with HUBECs results in a mean 6-fold increase in CD34+CD38 cells, an expansion that correlates closely with the 4.1-fold increase in SRCs that we have observed in this study (J.P.C. et al, manuscript in preparation). These results suggest that newly generated SRCs during 7-day HUBEC cultures are quite possibly CD34+CD38 cells. Cell-sorting studies of HUBEC-cultured populations should help determine which population is enriched for SRCs following culture and provide a more precise understanding of the effect of HUBEC culture on primitive ABM subsets.

There are several potential mechanisms through which HUBEC culture may have increased the SCID-repopulating capacity of adult ABM CD34+ cells. First, contact with HUBECs may have triggered self-renewal divisions within a subpopulation of primitive marrow cells, resulting in an absolute increase in SRCs. This result would be consistent with the single-hit Poisson model as it has been applied previously.12 Alternatively, exposure to HUBECs may have positively altered the engraftment capacity of the limited number of SCID-repopulating cells within the steady-state ABM CD34+ population. Alteration of adhesion receptor expression on primitive cells has been associated with enhanced engraftment in murine models,44,45 and the up-regulation of CXCR4 on CB cells during culture has been associated with increased engraftment in NOD/SCID mice.46 Engagement of the Jagged/Notch pathway also has been shown to promote the maintenance of primitive hematopoietic cells during culture.47 Finally, CD34 and CD34+CD38+ accessory cells contained within the HUBEC-cultured grafts may have facilitated the engraftment of SRCs within the recipient NOD/SCID marrow.48 49 However, since liquid suspension–cultured grafts contained equivalent numbers of CD34 and CD34+CD38+ accessory cells and failed to engraft NOD/SCID marrow, this explanation alone is incomplete. Whether HUBEC culture causes an absolute increase in ABM repopulating cells or augments the engraftment capacity of repopulating cells within adult bone marrow, the clinical impact of this method for stem cell expansion protocols would be the same: increased delivery of competent repopulating cells to the marrow.

The data from our noncontact HUBEC cultures with human ABM CD34+ cells suggest that cell-to-cell contact may not be required for the maintenance of marrow SRCs in this system. This result differs from previous studies, which have demonstrated a requirement for either stroma cell contact or adhesion via integrins to the fibronectin-COOH domain for the maintenance of adult-source stem cells during exposure to cytokines.50 51 In this study, noncontact HUBEC cultures maintained a percentage of CD34+CD38 cells at day 7, and more importantly, the progeny of these cultures maintained SCID-repopulating capacity. Although we did not perform a limiting dilution analysis to estimate the SRC frequency within noncontact HUBEC cultures, our results suggest a differential maintenance of SRCs within noncontact HUBEC cultures as compared with liquid suspension cultures. The lack of requirement for cell-to-cell contact may also be clinically advantageous since endothelial cell contamination of hematopoietic grafts would be eliminated.

Since the clinical transplantation of cord blood CD34+cells is limited by low cell numbers and delayed neutrophil/platelet engraftment,52 we are currently testing the capacity of HUBEC culture to expand repopulating cells within this population. Additionally, we will be performing serial transplantation studies to confirm that HUBEC coculture maintains cells with long-term repopulating capacity,53,54 and we plan to test HUBEC culture with other cytokine combinations, such as SCF plus flt-3 ligand plus thrombopoietin (TPO) plus IL-6/ soluble IL-6 receptor (sIL-6R),27 in order to further augment the expansion of SRCs presented here. Finally, the concentrations of IL-3 (5 ng/mL), IL-6 (5 ng/mL), and GM-CSF (2 ng/mL) that we used in this study were lower than other investigators have previously applied to induce stem cell proliferation in vitro.41 55 We will additionally test whether higher concentrations of these cytokines might further increase the SRC expansion observed here.

Although remarkable progress has been made recently in the ex vivo expansion of human cord blood SRCs, this progress has not translated into successful methods for the ex vivo expansion of either bone marrow– or peripheral blood–mobilized stem cells. The limiting dilution analysis presented here demonstrates that long-term repopulating cells within adult human bone marrow can be increased via exposure to human brain endothelial cells. This culture method may prove clinically useful for both the ex vivo expansion and genetic modification of adult human bone marrow stem cells.

The authors wish to acknowledge Dr David Venzon for providing the statistical analysis and for comments in the preparation of this manuscript.

Prepublished online as Blood First Edition Paper, August 1, 2002; DOI 10.1182/blood-2002-04-1238.

Supported by a grant (PE 0603706N) from the US Office of Naval Research; also supported in part by research funding from Large Scale Biology Corp to J.P.C. via the Cooperative Research and Development Agreement (No. NCRADA-NMRDC/NMRI/Biosource-97-588) between the Naval Medical Research Center and Large Scale Biology Corporation and NIH grant P01 CA70970.

The Johns Hopkins University holds patents on CD34 monoclonal antibodies and related inventions. C.C. is entitled to a share of the sales royalty received by the University under licensing agreements between the University, Becton Dickinson Corp, and Baxter HealthCare Corp. The terms of these arrangements have been reviewed and approved by the University in accordance with its conflict of interest policies.

J.P.C. and T.A.D. are currently employed by Large Scale Biology Corp whose potential product was studied in the present work.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Gan
OI
Murdoch
B
Larochelle
A
Dick
JE
Differential maintenance of primitive human SCID- repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells.
Blood.
90
1997
641
650
2
Nolta
JA
Smorgorzewska
EM
Kohn
DB
Analysis of optimal conditions for retroviral-mediated transduction of primitive human hematopoietic cells.
Blood.
86
1995
101
110
3
Gothot
A
van der Loo
J
Clapp
D
Srour
E
Cell cycle related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in non-obese diabetic/severe combined immune-deficient mice.
Blood.
92
1998
2641
2649
4
Traycoff
C
Orazi
A
Ladd
A
Rice
S
McMahel
J
Srour
E
Proliferation-induced decline of primitive hematopoietic progenitor cell activity is coupled with an increase in apoptosis of ex vivo expanded CD34+ cells.
Exp Hematol.
26
1998
53
62
5
Mobest
D
Goan
S
Junghahn
I
et al
Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells.
Stem Cells.
17
1999
152
161
6
Williams
D
Ex vivo expansion of hematopoietic stem and progenitor cells: robbing Peter to pay Paul?
Blood.
81
1993
3169
3177
7
Larochelle
A
Vormoor
J
Hanenberg
H
et al
Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy.
Nat Med.
2
1996
1329
1337
8
Bhatia
M
Wang
J
Kapp
U
Bonnet
D
Dick
J
Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice.
Proc Natl Acad Sci U S A.
94
1997
5320
5325
9
Bhatia
M
Bonnet
D
Murdoch
B
Gan
O
Dick
JE
A newly discovered class of human hematopoietic cells with SCID-repopulating activity.
Nat Med.
4
1998
1038
1045
10
Cashman
J
Lapidot
T
Wang
J
et al
Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice.
Blood.
89
1997
4307
4316
11
Holyoake
T
Nicolini
F
Eaves
C
Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow.
Exp Hematol.
27
1999
1418
1427
12
Wang
JC
Doedens
M
Dick
JE
Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay.
Blood.
89
1997
3919
3924
13
Uchida
N
Fujisaki
T
Eaves
AC
Eaves
CJ
Transplantable hematopoietic stem cells in human fetal liver have a CD34+ side population (SP) phenotype.
J Clin Invest.
108
2001
1071
1077
14
Aiuti
A
Friedrich
C
Sieff
CA
Gutierrez-Ramos
JC
Identification of distinct elements of the stromal microenvironment that control human hematopoietic stem/progenitor cell growth and differentiation.
Exp Hematol.
26
1998
143
157
15
Dexter
TM
Allen
TD
Lajtha
LG
Conditions controlling the proliferation of hematopoietic stem cells in vitro.
J Cell Physiol.
91
1977
335
344
16
Dorshkind
K
Regulation of hemopoiesis by bone marrow stromal cells and their products.
Annu Rev Immunol.
8
1990
111
137
17
Verfaillie
CM
Direct contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long term in vitro hematopoiesis.
Blood.
79
1992
2821
2826
18
Rafii
S
Shapiro
F
Rimarachin
J
et al
Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion.
Blood.
84
1994
10
19
19
Rafii
S
Shapiro
F
Pettengell
R
et al
Human bone marrow microvascular endothelial cells support long term proliferation and differentiation of myeloid and megakaryocytic progenitors.
Blood.
86
1995
3353
3363
20
Lu
LS
Wang
SJ
Auerbach
R
In vitro and in vivo differentiation into B cells, T cells, and myeloid cells of primitive yolk sac hematopoietic precursor cells expanded > 100-fold by co-culture with a clonal yolk sac endothelial cell line.
Proc Natl Acad Sci U S A.
93
1996
14782
14787
21
Davis
TA
Robinson
DH
Lee
KP
Kessler
SW
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.
Blood.
85
1995
1751
1761
22
Chute
JP
Saini
AA
Kampen
RL
Wells
MR
Davis
TA
A comparative study of the cell cycle status and primitive cell adhesion molecule profile of human CD34+ cells cultured in stroma-free versus porcine microvascular endothelial cell cultures.
Exp Hematol.
27
1999
370
379
23
Brandt
JE
Galy
AH
Luens
KM
et al
Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line.
Exp Hematol.
26
1998
950
961
24
Brandt
JE
Bartholomew
A
Fortman
JD
et al
Ex vivo expansion of autologous bone marrow stem cells with porcine microvascular endothelial cells results in a graft capable of rescuing lethally irradiated baboons.
Blood.
94
1999
106
113
25
Piacibello
W
Sanavio
F
Severino
A
et al
Engraftment in non-obese diabetic severe combined immunodeficient mice of human CD34+ cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells.
Blood.
93
1999
3736
3749
26
Bhatia
M
Bonnet
D
Kapp
U
Wang
J
Murdoch
B
Dick
J
Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.
J Exp Med.
186
1997
619
624
27
Ueda
T
Tsuji
K
Yoshino
H
et al
Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor.
J Clin Invest.
105
2000
1013
1021
28
Conneally
E
Cashman
J
Petzer
A
Eaves
C
Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice.
Proc Natl Acad Sci U S A.
94
1997
9836
9841
29
Jordan
CT
Yamasaki
G
Minamoto
D
High-resolution cell cycle analysis of defined phenotypic subsets within primitive human hematopoietic cell populations.
Exp Hematol.
24
1996
1347
1355
30
Schulz
LD
Schweitzer
P
Christianson
S
et al
Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.
J Immunol.
154
1995
180
191
31
Trischmann
TM
Schepers
KG
Civin
CI
Measurement of CD34+ cells in bone marrow by flow cytometry.
J Hematother.
2
1993
305
313
32
Taswell
C
Limiting dilution assays for the determination of immunocompetent cell frequencies, I: data analysis.
J Immunol.
126
1981
1614
1619
33
Horwitz
M
Uses and growth of hematopoietic cell transplantation.
Hematopoietic Cell Transplantation.
2nd ed.
Thomas
ED
Blume
KG
Forman
SJ
1998
12
18
Oxford University Press
Malden, MA
34
Ciancio
G
Garcia-Morales
R
Mathew
J
et al
Donor bone marrow infusions are tolerogenic in human renal transplantation.
Transplant Proc.
33
2001
1295
1296
35
Orlic
D
Girard
L
Jordan
C
et al
The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.
Proc Natl Acad Sci U S A.
93
1996
11097
11102
36
Chute
J
Saini
A
Wells
M
et al
Preincubation with endothelial cell monolayers increases gene transfer efficiency into human bone marrow CD34+CD38− progenitor cells.
Hum Gene Ther.
11
2000
2515
2528
37
Van der Sluijs
JP
van den Bos
C
Baert
MR
van Beurden
CA
Ploemacher
RE
Loss of long term repopulating ability in long-term bone marrow culture.
Leukemia.
7
1993
725
732
38
Dorrell
C
Gan
O
Pereira
D
Hawley
R
Dick
J
Expansion of human cord blood CD34+CD38− cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function.
Blood.
95
2000
102
110
39
Mehta
K
McQueen
T
Manshouri
T
Andreef
M
Collins
S
Albitar
M
Involvement of retinoic acid receptor-alpha-mediated signaling pathway in induction of CD34 cell-surface antigen.
Blood.
89
1997
3607
3614
40
Wang
L
Liu
H
Xia
Z
Broxmeyer
H
Lu
L
Expression and activation of caspase-3/CPP32 in CD34+ cord blood cells is linked to apoptosis after growth factor withdrawal.
Exp Hematol.
28
2000
907
915
41
Glimm
H
Oh
I-H
Eaves
C
Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0.
Blood.
96
2000
4185
4193
42
Peters
S
Kittler
E
Ramshaw
H
Quesenberry
P
Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts.
Blood.
87
1996
30
37
43
Van Hennick
P
de Koning
A
Ploemacher
R
Seeding efficiency of primitive human hematopoietic cells in non-obese diabetic/severe combined immune deficiency mice: implications for stem cell frequency assessment.
Blood.
94
1999
3055
3061
44
Wagers
A
Allsopp
R
Weissman
I
Changes in integrin expression are associated with altered homing properties of Lin(−/lo)Thy1.1(lo)Sca-1(+)c-kit(+) hematopoietic stem cells following mobilization by cyclophosphamide/granulocyte colony-stimulating factor.
Exp Hematol.
30
2002
176
185
45
Berrios
V
Dooner
G
Nowakowski
G
et al
The molecular basis for the cytokine-induced defect in homing and engraftment of hematopoietic stem cells.
Exp Hematol.
29
2001
1326
1335
46
Peled
A
Petit
I
Kollet
O
et al
Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.
Science.
283
1999
845
848
47
Varnum-Finney
B
Xu
L
Brashem-Stein
C
et al
Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch 1 signaling.
Nat Med.
6
2000
1278
1281
48
Bonnet
D
Bhatia
M
Wang
J
Kapp
U
Dick
J
Cytokine treatment or accessory cells are required to initiate engraftment of purified primitive human hematopoietic cells transplanted at limiting doses into NOD/SCID mice.
Bone Marrow Transplant.
23
1999
203
209
49
Verstegen
M
van Hennik
P
Terpstra
W
et al
Transplantation of human umbilical cord blood cells in macrophage-depleted SCID mice: evidence for accessory cell involvement in expansion of immature CD34+CD38− cells.
Blood.
91
1998
1966
1976
50
Breems
D
Blokland
E
Siebel
K
Mayen
A
Engels
L
Ploemacher
R
Stroma-contact prevents loss of hematopoietic stem cell quality during ex vivo expansion of CD34+ mobilized peripheral blood stem cells.
Blood.
91
1998
111
117
51
Dao
M
Hashino
K
Kato
I
Nolta
J
Adhesion to fibronectin maintains regenerative capacity during ex vivo culture and transduction of human hematopoietic stem and progenitor cells.
Blood.
92
1998
4612
4621
52
Thomson
B
Robertson
K
Gowan
D
et al
Analysis of engraftment, graft-versus-host disease, and immune recovery following unrelated donor cord blood transplantation.
Blood.
96
2000
2703
2711
53
Hogan
C
Schpall
E
Keller
G
et al
Differential long-term and multilineage engraftment potential from subfractions of human CD34+ cord blood cells transplanted into NOD/SCID mice.
Proc Natl Acad Sci U S A.
99
2002
413
418
54
Lewis
I
Almeida-Porada
G
Du
J
et al
Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system.
Blood.
97
2001
3441
3449
55
Glimm
H
Eaves
C
Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture.
Blood.
94
1999
2161
2168

Author notes

John P. Chute, Large Scale Biology Corporation, 3333 Vaca Valley Pkwy, Vacaville, CA 95688; e-mail:john.chute@lsbc.com.

Sign in via your Institution