SEARCH

SEARCH BY CITATION

Keywords:

  • cord blood;
  • CD34+ purification;
  • colony-forming cells;
  • LTC-IC;
  • expansion

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

In-vitro expansion of human cord blood (CB) cells could enhance peripheral blood recovery and ensure long-term engraftment of larger recipients in the clinical transplant setting. Enrichment of CD34+ cells using the MiniMACS column has been evaluated for the preparation of CB CD34+ cells before and after expansion culture. Repurification of CD34+ cells after culture would assist accurate phenotypic and functional analysis. When fresh CB mononuclear cells (MNC) were separated, the MACS positive (CD34+) fraction (90.1% pure) contained a mean (± SD, n = 5) of 93.0 ± 8.0% of the eluted CD34+ cells, 99.6 ± 0.7% of the CFU-GM and all of the eluted long-term culture-initiating cells (LTC-IC). Cord blood CD34+ cells were then cultured for 14 d with IL-3, IL-6, SCF, G-CSF and GM-CSF, each at 10 ng/ml. The total cell expansion was 2490 ± 200-fold and the CD34+ cell expansion was 49 ± 17-fold. The percentage of CD34+ cells present after expansion culture was 1.2 ± 0.85%. When these cells were repurified on the MiniMACS column, the MACS positive fraction only contained 40.3 ± 13.4% of the eluted CD34+ cells which was enriched for the mature CD34+ CD38+ subset, 24.4 ± 8.8% of the eluted CFU-GM and 79.5 ± 11.0% of the LTC-IC. The remaining cells were eluted in the MACS negative fraction. In conclusion, repurification of cultured CD34+ cells does not yield a representative population and many progenitors are lost in the MACS negative fraction. This can give misleading phenotypic and functional data. Cell losses may be important in the clinical setting if cultured cells were repurified for purging.

Cord blood has been used as a source of haemopoietic stem cells for transplantation since 1989 (Gluckman et al, 1989) to treat a wide variety of malignant and non-malignant haemopoietic disorders (Wagner et al, 1995; Kurtzberg et al, 1996). Despite limited progenitor numbers, sufficient marrow repopulating cells are present, because sustained engraftment in children has been documented (Gluckman et al, 1989). However, the number of nucleated cells infused per kilogram is a major factor in the recovery of neutrophil and platelet counts (Gluckman et al, 1997). We hypothesized that expansion of post-progenitor cells, progenitor cells and primitive cells such as LTC-IC may be required for adequate engraftment in larger recipients. For these reasons we have investigated the in vitro expansion of CB cells.

Enriched CD34+ cells are routinely used as starting material for expansion cultures. We have evaluated the MiniMACS immunomagnetic separation column for the preparation of CD34+ cells prior to and after expansion culture. Enrichment of CD34+ cells from cultures, if successful, would enable accurate phenotypic analysis using a population of cells of similar purity to the starting material.

In this study we enriched CB CD34+ cells using the MiniMACS column. Its performance was assessed by monitoring the positive (CD34+) and negative fractions for cell recovery and the presence of colony forming cells (CFC) and LTC-IC. Enriched CD34+ cells from the MACS positive fraction were then cultured for 14 d in the presence of IL-3, IL-6, G-CSF, GM-CSF and SCF. Expansion of total cells, CFU-GM and LTC-IC was calculated, together with flow cytometric analysis, to estimate the expression of CD38 and HLA-DR in cultured cells. Cells from expansion cultures were also reapplied to the miniMACS column and the performance of the cells on the column investigated as before.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Cord blood collection

Umbilical CB samples were collected by gravity into sterile 50 ml tubes containing 1000 i.u. heparin after the umbilical cord had been clamped and cut by a midwife. All samples were from normal full-term deliveries. Cord blood collection had local hospital ethics committee approval and time of clamping of the umbilical cord and collection of CB was entirely at the discretion of the midwife.

Purification of CD34+ cells from fresh cord blood

Cord blood MNC were prepared from 40–50 ml CB using Ficoll-Hypaque (Lymphoprep d = 1.077 g/ml, Nycomed, Birmingham) density gradient separation followed by treatment with ammonium chloride lysing solution to remove red cells. Cells were then suspended in cold phosphate buffered saline containing 0.5% bovine serum albumin (BSA) and 5 mm EDTA (Magnetic cell sorting ‘MACS’ buffer) and incubated for 15 min with anti-CD34 antibody (QBEnd 10) and human IgG to prevent non-specific binding. After washing with MACS buffer the cells were incubated for 15 min with colloidal superparamagnetic MACS microbeads recognizing QBEnd 10. Cells were loaded onto a MiniMACS column (Miltenyi Biotec Ltd, Bisley) held within a high-density magnetic field to retain CD34+ cells. Unbound cells (CD34 cells) were eluted with MACS buffer and retained CD34+ cells were eluted by washing with buffer after the column had been removed from the magnet. Cells in the MACS positive fraction were applied to a second column and the purification step repeated. Mononuclear cells and cells from the MACS positive and negative fractions were assessed by cell counting, flow cytometry, clonogenic assay and LTC-IC/cobblestone area forming cells (CAFC).

Expansion cultures

Enriched CD34+ cells (5 × 103 /ml) were cultured at 37°C for 14 d in 25 cm2 vented flasks (Falcon 3108, obtained from Fahrenheit, Milton Keynes) in 10 ml Iscove's modified Dulbecco's medium (IMDM, 300 mOsmol/kg H2O, Sigma I7633), containing 20% fetal calf serum (FCS, Stem Cell Technologies, Vancouver HCC-6450) and growth factors IL-3, IL-6, SCF, G-CSF and GM-CSF (gifts from Amgen), all at 10 ng/ml. On days 7 and 10, half the media and cells from each flask were transferred to a new flask and additional media added to all flasks to maintain a volume of 10 ml.

At the end of the culture period, cells were harvested by centrifugation for 10 min at 600 g followed by resupension in 8–10 ml IMDM. Small aliquots were removed for cell counting, flow cytometry, clonogenic assay and LTC-IC/CAFC. All the remaining cells (approx. 85%) were resuspended in MACS buffer for incubation with QBEnd 10 and immunomagnetic purification as above.

Flow cytometry

Cord blood MNC and cells in the MACS positive and negative fractions were assessed for CD34+ content by dual-labelling with FITC-conjugated anti-CD45 (clone T29/33 DAKO, High Wycombe) and a PE-conjugated anti-CD34 (clone HPCA-2, Beckton Dickinson, Cowley) or an appropriate PE-conjugated control. Samples were examined on a Coulter Epics XL flow cytometer and data were analysed using the Epics Listmode software. Initial gating on CD45+ events was used to exclude debris and red cells. Only CD34+, CD45+ events with low side scatter were counted as CD34+. Cells from expansion cultures, before and after MACS purification, were analysed using three-colour flow cytometry. Cells were labelled with PE-conjugated anti-CD34, FITC-conjugated anti-CD38 (clone HIT2, Caltag, supplied by Bradsure Biologicals, Loughborough) and Tricolour-conjugated anti-HLA-DR (clone TU 36, Caltag). Two control samples were used. Cells in the first control were labelled with PE-conjugated anti-CD34 and isotype controls for FITC and Tricolour. The second control was labelled with appropriate isotype controls for all three markers PE, FITC and Tricolour. CD34+ events were identified by comparing plots of PE-IgG/side scatter (SS) and PE-CD34/SS. Markers were then set for the isotype FITC and Tricolour controls so that more than 95% of the CD34+ events gated were in the negative quadrant. This method avoided inclusion of CD34 events in the controls.

Assay for haemopoietic colony forming cells

Haemopoietic CFCs were assayed as described previously (Denning-Kendall et al, 1996) using 1.3% methylcellulose in IMDM containing 10% 5637 conditioned medium, 10% BSA (Stem Cell Technologies HCC-9300), 30% FCS (HCC-6150) and 2 U/ml EPO. Triplicate cultures were plated in 0.25 ml volumes in 24-well tissue culture plates (Falcon 3047). Mononuclear cells or cells from expansion cultures were plated at 104 cells/well, MACS positive cells at 125–250 cells/well. MACS negative cells from fresh CB at 18–28 ×104 cells/well and MACS negative cells from expansion cultures at 104 cells/well.

Assessment of LTC-IC and CAFC

The frequency of week 5 LTC-IC and CAFC were determined using a limiting dilution assay as previously described (Nicol et al, 1996). Briefly, cells of interest were cultured with cryopreserved irradiated bone marrow feeder cells in 96-well plates at six different concentrations with 24 replicates per dilution. Mononuclear cells were plated at concentrations ranging from 250 to 8000 cells/well, MACS positive cells 25–800 cells/well, MACS negative cells (from both fresh and cultured CB) 1000–32 000 cells/well and cells from expansion cultures 4000–128 000 cells/well in 0.1 ml IMDM (340 mOsmol/kg containing 10% fetal calf serum, 10% horse serum and 5 × 10−7 m hydrocortisone). Plates were incubated at 33°C and fed by half media changes every 10 d.

For assessment of CAFC after 5 weeks of culture all wells were scored microscopically for the presence of 12 or more closely associated, small, embedded cells. To estimate the LTC-IC frequency, three dilutions which had >15% CAFC positive wells were chosen and the non-adherent and adherent cells were harvested from these wells by the addition of 0.25% trypsin. The cells from each well were assayed for CFC by incubation in 1 ml methylcellulose mixture incubated in 35 mm dishes (Stem Cell Technologies) chosen for their inability to support growth of anchorage-dependent cells. Using this method, stroma does not reform on the base of the dish and obscure or inhibit colony formation. After 14 d incubation, dishes were scored as positive or negative for the presence of colonies. On the assumption that week 5 CFC must have arisen from an LTC-IC, frequencies were calculated with the Poisson formula from the percentage of negative wells using the Strijbosch computer program (Strijbosch et al, 1987).

Statistics

All results are reported as mean ± standard deviation (SD). Correlation coefficients were calculated using linear regression analysis.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Cord blood mononuclear cells

Processing 40–50 ml of CB using Lymphoprep routinely provided 2.0 ± 0.5 × 108 MNC (n = 5), which contained 1.42 ± 1.2% CD34+ cells. Their average CFU-GM cloning efficiency was 22.0% ( or 220 ± 90 CFU-GM/105 MNC). The cloning efficiency of CD34+ cells for total CFU (CFU-GM and BFU-E) was 52.5 ± 26.1%. No CFU-Mix were seen under our assay conditions. The mean LTC-IC frequency per MNC was 1/1425 and this corresponded to 1/21(range 1/16–1/47) of the CD34+ cells being a week 5 LTC-IC (Table I).

Table 1. Table I. Properties of cord blood cells applied to MiniMACS columns (n = 5). ND = not determined.Thumbnail image of

MiniMACS CD34+ cells from fresh cord blood

The mean purity of cord blood CD34+ cells following MiniMACS separation of CB MNC was 90.1 ± 5.4%. The MiniMACS column proved to be very efficient in separating primitive haemopoietic cells from more mature cells with >90% of the eluted CD34+ cells, CFU-GM and LTC-IC being present in the MACS positive fraction (Fig 1). CD34+ cells were found in the negative fraction on some occasions (4/7 samples), where they accounted for 12.3 ± 6.3% of the eluted CD34+ cells. However, they gave rise to <1% of the eluted CFU-GM, producing mainly small BFU-E (15 ± 6 BFU-E/105 cells). In addition, application of MNC to the column resulted in very little cell loss with good recovery of nucleated cells and progenitors (Table II). Analysis of cells pre- and post-MiniMACS column showed that the purification process was not associated with loss in clonogenicity or LTC-IC activity of CD34+ cells. The correlation for CFU-GM/CD34+ and BFU-E/CD34+ cell before and after column separation for individual samples was 0.93 and 0.81 respectively (n = 7). The mean LTC-IC frequency/ CD34+ cell in the MACS positive fraction was 1/24. This was no different to the mean LTC-IC frequency/CD34+ cell (1/21) in the MNC fraction applied to the column. The correlation of LTC-IC frequencies for individual samples before and after column separation was 0.96.

image

Figure 1. . Elution of CD34+ cells, CFU-GM and LTC-IC in the MACS positive fraction from fresh cord blood MNC and cultured cord blood CD34+ cells (n = 5). Percentage yield is number of cells eluted in positive fraction divided by total number of cells eluted ×100. The figure demonstrates the very low yields of CD34+ cells and CFU-GM from cultured cells.

Download figure to PowerPoint

Table 2. Table II. Total recovery of cells and progenitors after MiniMACS separation (%) (n = 5).Thumbnail image of

CD34+ cells in expansion culture

Total cell numbers increased 2490 ± 200-fold during expansion culture. The expansion of CD34+ cells and CFU-GM was not as great as the increase in total cells but was substantial (49 ± 17-fold) with a close correlation between the expansion of CD34+ cells and CFU-GM for individual samples. The percentage of CD34+ cells in the cultures fell from around 90% to 1.2 ± 0.85% after 14 d expansion and the proportion of cells which were CFU-GM fell from 154/103 cells to 3/103, indicating that mature cells were generated faster than progenitor cells. Expansion of LTC-IC and CAFC was small but consistent (2.2–5.3-fold and 1.2–3.7-fold respectively).

Comparison of the behaviour of fresh and cultured CB CD34+ cells on the MiniMACS column

The nature of the expansion meant that the number of cells present at the end of the experiment and the proportion of progenitors present was very similar to fresh CB MNC with the exception of a reduced frequency of primitive LTC-IC/CAFC (Table I). Application of cultured CD34+ cells to the miniMACS column resulted in very little cell loss, with high percentages of nucleated cells and progenitors being eluted from the column. 2Table II shows the total recovery (yield in the positive plus negative fractions) from five experiments.

However, when cultured CB CD34+ cells were applied to the miniMACS column for CD34+ enrichment, they behaved very differently to fresh CB cells. Although the cells in the MACS positive fraction were 82.5 ± 11.7% CD34+, this fraction only contained 40.3% of the eluted CD34+ cells, 24.4% of the eluted CFU-GM and 79.5% of the eluted LTC-IC (Fig 1). The remaining cells of each type were eluted in the MACS negative fraction. The CD34+ cells in the MACS negative fraction were enriched for colony forming activity compared with those in the MACS positive fraction and hence accounted for nearly 86% of eluted CFU-GM (Fig 2). Conversely, the CD34+ cells MACS positive fraction were enriched for LTC-IC activity compared to those eluted in the MACS negative fraction (Fig 3).

image

Figure 2. . Comparison of the clonogenicity (percentage of CD34+ cells which are CFU-GM) of cultured cord blood CD34+ cells before application to the miniMACS column and those in the MACS positive and negative fractions after column separation. More CFU-GM were associated with cells in the negative fraction (n = 3).When fresh CB MNC were fractionated on the miniMACS, only 0.1% of the MACS negative fraction cells were CD34+ and their clonogenicity was only 0.93%.

Download figure to PowerPoint

image

Figure 3. . Comparison of the distribution of LTC-IC during separation of cultured CD34+ cells on the miniMACS colmun. The results show that CD34+ cells which were LTC-IC were concentrated in the MACS positive fraction (n = 3).

Download figure to PowerPoint

Phenotypic analysis of MiniMACS purified cultured cells by flow cytometry to assess the proportion of cells carrying CD38 or HLA-DR was achieved by the analysis of a minimum of 10 000 CD34+ events. The MACS positive CD34+ cells were >85% CD38+/ DR+ (Fig 4) with very few CD38 cells. Such accurate flow cytometric analysis of CD34+ cells after culture before miniMACS separation was not possible because fewer CD34+ events were available, but using the same gating and running both samples on the same day it was clear that CD34+ cells in the unfractionated cultured population contained a much higher proportion of CD38 cells.

image

Figure 4. . Percentage of each CD34+ phenotype on cultured CD34+ cells before application to the MiniMACS column and those in the MACS positive fraction after column separation. In each case the total is 100% (n = 4).

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Enriched CD34+ CB cells harvested in the MACS positive fraction had the same clonogenic potential and LTC-IC frequency as the MNC CD34+ population, indicating that they were representive of the CD34+ population found in whole CB. The column processing was also very efficient in that very few CD34+ cells or progenitors were lost either on the column or to the negative fraction. This is in agreement with other studies which conclude that the miniMACS system is superior to CEPRATE avidin-biotin column separation or Dynabeads in terms of purity and yield (De Wynter et al, 1995; Laver et al, 1995).

When CB CD34+ cells were cultured in suspension in the presence of growth factors a substantial expansion of CB progenitors and a small but consistent expansion of 5-week LTC-IC/CAFC was observed. During the culture mature cells were produced at a faster rate than progenitors or LTC-IC so that the resulting percentage of CD34+ cells present and the proportion of cells which were CFU-GM was very similar to the CB MNC fraction. However, when these cells were applied to the MiniMACS column substantial numbers of progenitors and LTC-IC were eluted in the MACS negative fraction. Cells eluted in the positive fraction were highly enriched for the mature CD38+/DR+ subpopulation and were not representative of the cell population applied to the column. If cultured CD34+ cells were enriched by the MiniMACS column for analysis there would be an under estimation of the expansion of CFU-GM and LTC-IC together with misleading phenotypic and functional data.

The CD34 molecule expressed on haemopoietic progenitor cells contains a large number of epitopes.These epitopes can be categorized according to their sensitivity to degredation by neuraminidase, chymopapain and a glycoprotease from Pasteurella haemolytica (Sutherland et al, 1992). Class I epitope is sensitive to all three enzymes, class II, neuraminidase resistant, glycoprotease/chymopapain sensitive and class III is resistant to all three enzymes. Cleavage of the 110 kD CD34 structure by the glycoprotease genereates a cell-bound protein of 75 kD, identified by class III antibodies such as HPCA-2 used for flow cytometric analysis of all our samples. However, the glycoprotease removes the epitope recognized by QBEnd 10, the antibody used in the enrichment of CD34+ cells on the immunomagnetic column. Therefore it is possible for differential epitope expression on haemopoietic cells, which may be related to their maturation or function (Steen et al, 1996), such that some may bind QBEnd 10 and HPCA-2 but some may only bind HPCA-2.

On the immunomagnetic column separation of cells into the MACS positive or negative fraction depended on the binding of QBEnd 10 and detection of CD34+ cells in either fraction used the class III antibody HPCA-2 (which should detect all CD34+ cells). Our experience with fresh CB CD34+ cells suggests that a very high proportion express both class II and class III epitopes. The negative fraction contained at the most 19.5% of the eluted CD34+ cells, but accounted for <2% of eluted CFU-GM, suggesting that these were very mature progenitors. Data of Steen et al (1996) would support this theory, as CD34+ cells detected by HPCA-2 and not QBEnd 10 have high forward scatter consistent with slightly granular cytoplasm.

Analysis of MACS fractions from cultured CD34+ cells showed not only that a much higher proportion of cells did not bind HPCA-2 and were eluted in the MACS negative fraction, but in contrast to fresh CB CD34+ cells, lack of class II epitope was not associated with lack of clonogenicity or LTC-IC activity. Indeed, the MACS negative fraction was enriched for CFU-GM. The CD34+ cells which seemed to stain most strongly with HPCA-2 were CD38+/DR+, and although strict comparison with cultured cells before MiniMACS separation is difficult, it seems that this subpopulation was enriched in the positive fraction (Fig 4). Freshly isolated CB CD34+ cells are quiescent with 70–97% in G0/G1 (Traycoff et al, 1994; Hao et al, 1995; Engelhardt et al, 1997). During exposure to cytokines cell division increases so that after 72 h 40–50% are in cell cycle (Engelhardt et al, 1997) and after 7 d >95% of cells have divided (Traycoff et al, 1995). It is possible that rapidly dividing cells experience an early down-regulation of CD34 class I and II epitope expression out of sychrony with their functional development. This view is supported by the loss of more mature CFC to the MACS negative fraction whereas most of the LTC-IC were retained in the MACS positive fraction. There is also evidence that the CD38 antigen may be lost from CD38 positive cells during expansion culture, leading to falsely high estimates of CD38 cell expansion (Dorrell et al, 1998).

In addition, impaired engraftment of cultured cells has been reported (Brandt et al, 1998; Habibian et al, 1998; Gothot et al, 1998), which may also be due to changes in cell surface antigens induced by in vitro culture. These changes may be a reversible plastic feature correlated with cell cycle progression (Habibian et al, 1998), including decreased expression of adhesion proteins during late S/G2 (Becker et al, 1997) or an increase in peptides which allow cells to undergo endothelial transmigration so that infusion of cells in cycle would lead to the homing of cells in non-haemopoietic organs (Yong et al, 1998).

In conclusion, immunomagnetic columns are ideal for the production of enriched fresh CD34+ cells, but repurification of cultured CD34+ cells by this method does not yield a representative population, with many progenitors being lost in the negative fraction. This can give misleading phenotypic and functional data. These problems may be overcome in the research setting by the use of flowcytometric cell sorting or by using negative selection. In a clinical setting, repurification of CD34+ cells by positive selection on an immunomagnetic column of expansion cultures would lead to large cell losses. It is important that there is careful selection of CD34-specific antibodies for both cell purification and enumeration. Possible abnormal expression of cell surface antigens during processing and culture procedures should also be taken into account as this can alter their behaviour in vitro and in vivo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We thank the midwives in the central delivery suite Southmead Hospital for UCB collection and The London Clinic for supply of normal bone marrow samples for LTC-IC/CAFC assays. This work was funded by the Leukaemia Research Fund with additional funds from the Southmead Stem Cell Research Fund.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
  • 1
    Becker, P.S., Berrios, V., Nilsson, S.K., Dooner, N.B., Ramshaw, H.S. & Quesenberry, P.J. (1997) Stimulatory cytokines modulate adhesion receptor expression by murine LIN SCA+ cells and simultaneous blocking of α1, α4, and L-selectin inhibits hematopoietic progenitor stromal interactions. (Abstract). Blood, 90, 486a.
  • 2
    Brandt, J.E., Bartholomew, A.M., Nelson, M.C., Chute, J.P., Turian, J.V., Chen, L. & Hoffman, R. (1998) Rescue of lethally myeloablated baboons with ex vivo expanded grafts results in delayed engraftment. (Abstract). Experimental Hematology, 26, 699.
  • 3
    Denning-Kendall, P.A., Donaldson, C., Nicol, A., Bradle, B. & Hows, J. (1996) Optimal processing of human umbilical cord blood for clinical banking. Experimental Hematology, 24, 13941401.
  • 4
    De Wynter, E.A., Coutinho, L.H., Pei, X., Marsh, J.C.W., Hows, J., Luft, T. & Testa, N.G. (1995) Comparison of purity and enrichment of CD34+ cells from bone marrow, umbilical cord and peripheral blood (primed for apheresis) using five separation systems. Stem Cells, 13, 524532.
  • 5
    Dorrell, C., Gan, O.I., Pereira, D.S. & Dick, J.E. (1998) Expansion of human cord blood CD34+CD38 cells in ex vivo culture during retroviral transduction without a corresponding increase in SRC frequency: dissociation of SRC phenotype and function. (Abstract). Experimental Hematology, 2, 688.
  • 6
    Engelhardt, M., Kumar, R., Albanell, J., Pettengell, R., Han, W. & Moore, M. (1997) Telomerase regulation, cell cycle, and telomerase stability in primitive hematopoietic cells. Blood, 90, 182193.
  • 7
    Gluckman, E., Broxmeyer, H.E., Auerbach, A.D., Friedman, H.S., Douglas, G.W., Devergie, A., Esperou, H., Thierry, D., Socie, G., Lehn, P., Cooper, S., English, D., Kurtzberg, J., Bard, J. & Boyse, E.A. (1989) Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical cord blood from an HLA-identical sibling. New England Journal of Medicine, 321, 11741178.
  • 8
    Gluckman, E., Rocha, V., Boyer-Chammard, A., Locatelli, F., Arcese, W., Pasquini, R., Ortega, J., Souillet, G., Ferreira, E., Laporte, J-P., Fernadez, M. & Chastang, C. (1997) Outcome of cord-blood transplantation from related and unrelated donors. New England Journal of Medicine, 337, 373381.DOI: 10.1056/NEJM199708073370602
  • 9
    Gothot, A., Van Der Loo, J.C.M., Wade Clapp, D. & Srour, E.F. (1998) 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, 26412649.
  • 10
    Habibian, H.K., Peters, S.O., Hsieh, C.C., Wuu, J., Vergilis, K., Grimaldi, C.I., Reilly, J., Carlson, J.E., Frimberger, A.E., Stewart, F.M. & Quesenberry, P.J. (1998) The fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit. Journal of Experimental Medicine, 188, 393398.DOI: 10.1084/jem.188.2.393
  • 11
    Hao, Q-L., Shah, A.J., Thiemann, F.T., Smogorzewska, E.M. & Crooks, G.M. (1995) A functional comparison of CD34+CD38 cells in cord blood and bone marrow. Blood, 86, 37453753.
  • 12
    Kurtzberg, J., Laughlin, M., Graham, M.L., Smith, C., Olson, J.F., Halperin, E.C., Ciocci, G., Carrier, C., Stevens, C.E. & Rubinstein, P. (1996) Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. New England Journal of Medicine, 335, 157166.DOI: 10.1056/NEJM199607183350303
  • 13
    Laver, J., Traycoff, C.M., Abdel-Mageed, A., Gee, A., Lee, C., Turner, C., Srour, E.F. & Abboud, M. (1995) Effects of CD34+ selection and T cell immunodepletion on cord blood hematopoeitic progenitors: relevance to stem cell transplantation. Experimental Hematology, 23, 14921496.
  • 14
    Nicol, A., Nieda, M., Donaldson, C., Denning-Kendall, P., Bradley, B. & Hows, J.M. (1996) Cryopreserved human bone marrow stroma is fully functional in vitro. British Journal of Haematology, 94, 258265.DOI: 10.1046/j.1365-2141.1996.d01-1812.x
  • 15
    Steen, R., Tjonnfgjord, G.E., Gaudernack, G., Brinch, L. & Egeland, T. (1996) Differences in the distribution of CD34 epitopes on normal haemopoietic progenitor cells and leukaemic blasts. British Journal of Haematology, 9, 597605.DOI: 10.1046/j.1365-2141.1996.7052322.x
  • 16
    Strijbosch, L.W.G., Buurman, W.A., Does, R.J.M.M., Zinken, P.H. & Groenewegen, G. (1987) Limiting dilution assays, experimental design and statistical analysis. Journal of Immunological Methods, 97, 133140.DOI: 10.1016/0022-1759(87)90115-3
  • 17
    Sutherland, D.R., Marsh, J.C.W., Davidson, J., Baker, M.A., Keating, A. & Mellors, A. (1992) Differential sensitivity of CD34 epitopes to cleavage by Pasteurella haemolytica glycoprotease: implications for purification of CD34-positive progenitor cells. Experimental Hematology, 20, 590599.
  • 18
    Traycoff, C.M., Abboud, M.R., Laver, J., Wade, Clapp, D. & Srour, E.F. (1994) Rapid exit from G0/G1 phases of cell cycle in response to stem cell factor confers on umbilical cord blood CD34+ cells an enhanced ex vivo expansion potential. Experimental Hematology, 22, 12641272.
  • 19
    Traycoff, C.M., Kosak, S.T., Grigsby, S. & Srour, E.F. (1995) Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis. Blood, 85, 20592068.
  • 20
    Wagner, J.E., Kernan, N.A., Steinbuch, M., Broxmeyer, H.E. & Gluckman, E. (1995) Allogeneic sibling umbilical-cord blood transplantation in children with malignant and non-malignant disease. Lancet, 346, 214219.DOI: 10.1016/S0140-6736(95)91268-1
  • 21
    Yong, K.L., Watts, M., Thomas, N.S., Sullivan, A., Ings, S. & Linch, D. (1998) Transmigration of CD34+ cells across specialized and nonspecialized endothelium requires prior activation by growth factors and is mediated by PECAM-1 (CD31). Blood, 91, 11961205.