Mesenchymal stem cells from CD34(-) human umbilical cord blood.

Umbilical cord blood (UCB) is well known to be a rich source of stem cells especially for haematopoietic stem cells (HSCs). Recently, mesenchymal stem cells (MSCs) have also been shown to exist in cord blood. Although MSCs have been described by a subset of surface antigens after expansion, little is known about the cell surface phenotype of undifferentiated MSCs. The aim of this study therefore was to clarify whether undifferentiated MSCs are resident among CD34(-) UCB cells. CD34(+) cells were separated from UCB mononuclear cells (MNCs) by magnetic sorting and the CD34(-) cell fractions were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal calf serum (FCS) and basic-fibroblast growth factor. Isolated CD34(+) cells were also cultured in the same medium. Adherent fibroblast-like cells at passage 3-4 were analyzed by fluorescence-activated cell sorting (FACS) for MSC marker expression , and standard adipogenic, osteogenic and chondrogenic assays were used to investigate their differentiation potentials. After 4-5 weeks in culture, the cells from the CD34(-) fraction became confluent with flat and fibroblast-like morphology. These cells were positively stained for the mesenchymal cell markers CD29, CD73 and CD105. In adipogenic differentiation, the cells showed oil red O positive and expressed FABP4, adipsin and proliferation-activated receptor gamma-2 (PPARgamma2 genes) associated with adipogenesis. In osteogenic differentiation, calcium accumulation and osteocalcin were detected. The cells grown in chondrogenic conditions were positively stained for human aggrecan and expressed collagen type II and Sox-9 genes. In contrast, cells from the CD34(+) fraction failed to generate any cells with MSC morphology under the same culture conditions. Our results showed that UCB contained MSCs which are only resident in the CD34(-) fraction. The MSCs could be induced to differentiate into at least three lineage cell types, adipocytes, osteoblasts and chondrocytes.

Haematopoietic repopulating activity in human cord blood CD133+ quiescent cells.

We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133(+)G(1) cells. Here, we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures, the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (P<0.001) throughout the culture period. Furthermore, a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.

Marker gene transfer into human haemopoietic cells using a herpesvirus saimiri-based vector.

Herpesvirus-based gene therapy vectors offer an attractive alternative to retroviral vectors because of their episomal nature and ability to accommodate large transgenes. Saimiriine herpesvirus 2 (HVS) is a prototypical gamma-2 herpesvirus that can latently infect numerous different cell types. A cosmid-generated HVS vector in which transforming genes have been deleted and the marker gene encoding enhanced green fluorescent protein (HVS-GFP) has been incorporated was evaluated for its potential to transduce CD34+ haemopoietic progenitors selected from cord blood. Expression of GFP could subsequently be readily detected in cells of the erythroid lineage in both CFU-GEMM assays and liquid differentiation cultures. These results confirm the potential of HVS as a candidate vector for gene therapy applications using primitive haemopoietic cells and suggest that it may be applicable to disorders affecting cells of the erythroid lineage.

What is the future for cord blood stem cells?

Stem and progenitor cells are present in cord blood at a high frequency making these cells a major target population for experimental and clinical studies. Over the past decade there has been considerable developments in cord blood research and transplantation but despite the rapid progress many problems remain. The initial hope that cord blood would be an alternative source of haemopoietic cells for transplantation has been tempered by the fact that there are insufficient cells in most cord blood collections to engraft an adult of average weight. In attempts to increase the cell number, a plethora of techniques for ex-vivo expansion have been developed.These techniques have also proved useful for gene therapy. As cord blood cells possess unique properties this allows them to be utilised as suitable vehicles for gene therapy and long-term engraftment of transduced cells has been achieved. Current work examining the nature of the stem cells present in this haematological source indicates that cord blood contains not only haemopoietic stem cells but also primitive non-haemopoietic cells with high proliferative and developmental potential. As attention focuses on stem cell biology and the controversies surrounding the potential use of embryonic stem cells in treatment of disease, the properties of stem cells from other sources including cord blood are being re-appraised. The purpose of this article is to review some of the current areas of work and highlight biological problems associated with the use of cord blood cells.

Cord blood G(0) CD34+ cells have a thousand-fold higher capacity for generating progenitors in vitro than G(1) CD34+ cells.

We examined the functional differences between G(0) and G(1) cord blood CD34+ cells for up to 24 weeks in serum-free suspension culture, containing Flt-3 ligand, thrombopoietin and stem cell factor. By week 24, there is more than a 1,000-fold difference in granulocyte, macrophage-colony-forming cells (GM-CFC) cumulative production between the two populations, with cultures initiated from G(0) demonstrating an amplification of 1.1 x 10(5)-1.8 x 10(6) of GM-CFC compared to 45-2.7 x 10(3) for the G(1) cells. Cells from the initial G(0) population are able to produce about 250-fold higher numbers of BFU-E than those from G(1) which translates to 3 x 10(3)-1.1 x 10(5)-fold expansion and 25-390-fold expansion for G(0) and G(1), respectively. This amplification of the progenitor cells is reflected in finding that a greater proportion of the progeny of the G(0) population are CD34+, resulting in a 600-fold expansion of CD34+ cells at week 8. As in other in vitro systems, total cell expansion is less discriminatory of stem cell behavior than progenitor cells, and there is no significant difference in total cell numbers between G(0) and G(1) cultures with a mean fold expansion of 2 x 10(7) at 24 weeks.

CCR1 chemokine receptor expression isolates erythroid from granulocyte-macrophage progenitors.

Simple methods that separate progenitor cells of different hemopoietic lineages would facilitate studies on lineage commitment and differentiation. We used an antibody specific for the chemokine receptor CCR1 to examine mononuclear cells isolated from cord blood samples. When CD34(+) cells were separated into CD34(+)CCR1(+) and CD34(+)CCR1(-) cells and plated in colony-forming assays, the granulocyte/macrophage progenitors were found almost exclusively in the CD34(+)CCR1(+) cells. In contrast, the CD34(+)CCR1(-) cells contained the majority of the erythroid progenitors. There was a highly significant difference (P<0.002) in the total percentage distribution of both granulocyte-macrophage colony-forming cells and erythroid burst-forming units between the two populations. This is the first report of separation of erythroid progenitors from granulocyte/macrophage progenitors using a chemokine receptor antibody in cord blood samples. These results suggest that at the clonogenic progenitor cell stage the expression of CCR1 might be lineage-specific. This method should prove useful for studies on erythroid progenitor and granulocyte/macrophage differentiation.

NOD/SCID repopulating cells but not LTC-IC are enriched in human CD34+ cells expressing the CCR1 chemokine receptor.

Human haemopoietic stem and progenitor cells may be distinguished by the pattern of cell surface markers they display. The cells defined as 'stem' cells are heterogeneous and lack specific markers for their detection. However, they may be identified in in vitro assays such as the long-term culture initiating cell (LTC-IC) and in transplant assays involving immunosuppressed NOD/SCID mice. It is still not clear to what extent, if any, these cell populations overlap. The chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) prolongs survival of LTC-IC in suspension cultures and we now show that in longterm bone marrow cultures (LTBMC) maintenance of haemopoiesis was significantly better from the CD34+ cells which possess MIP-1alpha receptors (P < 0.006). We examined one MIP-1alpha receptor, CCR1, which is present on CD34+ cells from haemopoietic tissues. In LTBMC the production of GM-CFC from CD34+CCR1- cells was significantly higher (P < 0.02) than that from CD34+CCR1+ cultures and the incidence of LTC-IC was 3- to 6-fold higher in the CD34+CCR1- cell fraction. In contrast, the cells responsible for high levels of engraftment in NOD/SCID mice were contained in the CD34+CCR1+ cell fraction. The CD34+CCR1+ cells engrafted to high levels in NOD/SCID and generated large numbers of progenitor cells. Therefore, we conclude that LTC-IC and SRC may be distinguished on the basis of expression of the chemokine receptor CCR1.

Interest of cord blood stem cells.

Interest in cord blood stem cells was raised because of the possibility, now realised, of their use in clinical transplantation. The availability of only limited numbers of stem cells in cord blood compared to bone marrow or peripheral blood apheresis after cell mobilisation, led to experimental approaches that first aimed to characterise and then manipulate the stem cells present in cord blood. Their phenotypical and functional characteristics are not identical to those of stem cells in the bone marrow or those cells mobilised into the circulation. The cells selected for phenotype plus Go status show the higher capacity to generate progenitor cells in vitro and will offer the opportunity for mechanistic studies of stem cell self-renewal and proliferation. Another important field of exploration is to investigate the capacity of stem cells in cord blood for differentiation to tissues other than haemopoietic and to establish whether haemopoietic and non-haemopoietic lineages originate in truly multipotential cells or in cells coexisting in cord blood, which have already been limited to differentiation into specific tissue.

Dendritic cells infected with recombinant fowlpox virus vectors are potent and long-acting stimulators of transgene-specific class I restricted T lymphocyte activity.

The identification of dendritic cells (DC) as the major antigen-presenting cell type of the immune system, combined with the development of procedures for their ex vivo culture, has opened possibilities for tumour immunotherapy based on the transfer of recombinant tumour antigens to DC. It is anticipated that the most effective type of response would be the stimulation of specific, MHC class I restricted cytotoxic T lymphocytes capable of recognising and destroying tumour cells. In order to make this approach possible, methods must be developed for the transfer of recombinant antigen to the DC in such a way that they will initiate an MHC class I restricted response. Here, we demonstrate that murine DC infected with a recombinant fowlpox virus (rFWPV) vector stimulate a powerful, MHC class I restricted response against a recombinant antigen. A rFWPV containing the OVA gene was constructed and used to infect the DC line DC2.4. The infected DC2.4 cells were found to stimulate the T-T cell hybridoma line RF33. 70, which responds specifically to the MHC class I restricted OVA peptide SIINFEKL. The stimulatory ability of the rFWPV-infected DC2.4 cells lasted for at least 72 h after infection and was eventually limited by proliferation of uninfected cells. By comparison, DC2.4 cells pulsed with synthetic SIINFEKL peptide stimulated RF33.70 well initially, but the stimulatory ability had declined to zero by 24 h after pulsing. FWPV infection of DC2.4 up-regulated MHC and costimulatory molecule expression. rFWPV was also found to infect both immature and mature human DC derived from cord blood CD34+ progenitors and express transgenes for up to 20 days after infection. We conclude that rFWPV shows promise as a vector for antigen gene transfer to DC in tumour immunotherapy protocols.

Multicentre European study comparing selection techniques for the isolation of CD34+ cells.

Primitive haemopoietic cells are required for studies in both the clinical and research fields and a number of systems have been developed to facilitate isolation of these haemopoietic cell populations. We have analysed the results from several European centres using positive selection of CD34+ cells from haemopoietic tissues (n = 110). Four selection techniques including immunoaffinity columns (Ceprate LC), immunomagnetic beads (Dynabeads, Baxter Isolex 50) and submicroscopic magnetic beads (MACS) were used and the selected CD34+ cells were assessed for purity, yield and enrichment of colony-forming cells (CFC). The mean purities for all samples ranged from 68.4-78.4% for MACS, 33.9-69.9% for Dynabeads, 46.9-66.8% for Ceprate LC and 43.2-65% for Baxter Isolex 50. Yields were variable with all techniques. On average CFC enrichment using the immunoaffinity columns was greater than that observed for the other systems. Some techniques appear to be problematic and may require further expertise to improve the results. Nevertheless, the study demonstrates that highly purified CD34+ cells can be isolated from various haemopoietic sources, though yield and CFC enrichment varies significantly depending on the technique selected. This extends our previous report indicating that not all selection methods generate similar results and that there are differences in the purity, number and colony-forming ability of the cells recovered.

Properties of peripheral blood and cord blood stem cells.

Mobilized peripheral blood and cord blood are used for transplantation in adults and children. Currently methods which assess the engraftment potential of these cells rely on nucleated cell count, clonogenic colony assays (GM-CFC) and CD34+ cell enumeration. However, data have accumulated which indicate that the cells responsible for short-term and long-term engraftment are different and may be identified by a variety of techniques, including immunophenotyping, in vitro and in vivo assays. There is also evidence that primitive cells in peripheral blood progenitor cell grafts and cord blood are heterogeneous, as cells with similar functional behaviour express different phenotypes. Despite intensive research, the isolation and identification of a homogeneous population of human stem cells is still elusive. Nevertheless, it is possible to obtain CD34+ subpopulations enriched in primitive cells with many of the properties expected of stem cells. Using these cell fractions, the cytokines that induce proliferation, amplification, differentiation and self-renewal are being defined in order to develop improved protocols for expansion of specific populations. From these studies a number of interesting facts have emerged. Certain growth factors frequently used for progenitor cell expansion and gene transduction studies also induce differentiation and impair long-term engraftment. Further, the cytokines required for progenitor cell expansion are probably different to those which favour expansion of the primitive cells, with both the cell cycle status of CD34+ cells as well as the implication of telomere shortening probably needing to be considered where ex vivo manipulation is contemplated.

CD34+AC133+ cells isolated from cord blood are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells and dendritic cell progenitors.

The AC133 antigen is a novel antigen selectively expressed on a subset of CD34+ cells in human fetal liver, bone marrow, and blood as demonstrated by flow cytometric analyses. In this study, we have further assessed the expression of AC133 on CD34+ cells in hemopoietic samples and found that there was a highly significant difference between normal bone marrow and cord blood versus aphereses (p <0.0001) but not between bone marrow and cord blood. Most of the clonogenic cells (67%) were contained in the CD34+AC133+ fraction. Compared with cultures of the CD34+AC133- cells, generation of progenitor cells in long-term culture on bone marrow stroma was consistently 10- to 100-fold higher in cultures initiated with CD34+AC133+ cells and was maintained for the 8-10 weeks of culture. Only the CD34+AC133+ cells were capable of repopulating NOD/SCID mice. Human cells were detectable as early as day 20, with increased levels (67%) apparent 40 days post-transplantation. Five thousand CD34+AC133+ cells engrafted about 20% of the mice, while no engraftment was observed in animals transplanted with up to 1.2 x 10(5) CD34+AC133- cells. The CD34+AC133+ population was also enriched (seven-fold) in dendritic cell precursors, and the dendritic cells generated were functionally active in a mixed lymphocyte reaction assay. AC133+ cells should be useful in the study of cellular and molecular mechanisms regulating primitive hemopoietic cells.

Expression of macrophage inflammatory protein-1 receptors in human CD34(+) hematopoietic cells and their modulation by tumor necrosis factor-alpha and interferon-gamma.

Macrophage inflammatory protein-1alpha (MIP-1alpha) can stimulate growth inhibitory and potent chemotactic functions in hematopoietic cells. To investigate whether the action of MIP-1alpha may be regulated at the cellular receptor level, we studied the expression and modulation of MIP-1alpha receptors on CD34(+) cells isolated from normal bone marrow (NBM), umbilical cord blood (CB), and leukapheresis products (LP). Expression of MIP-1alpha receptors on CD34(+) cells was analyzed by two-color flow cytometry using a biotinylated MIP-1alpha molecule. The mean percentage of LP CD34(+) cells expressing the MIP-1alpha receptors was 67.7 +/- 7.2% (mean +/- SEM; n = 22) as compared with 89.9 +/- 2.6% (n = 10) and 74.69 +/- 7.04% (n = 10) in CB and NBM, respectively (P = .4). The expression of the MIP-1alpha receptor subtypes on LP CD34(+) cells was studied by indirect immunofluorescence using specific antibodies for the detection of CCR-1, CCR-4, and CCR-5. Microscopical examination revealed a characteristic staining of the cytoplasmic cell membrane for all three receptor subtypes. Detailed analysis of two LP samples showed that 65.8%, 4.4%, and 30.5% of CD34(+) cells express CCR-1, CCR-4, and CCR-5, respectively. Culture of LP CD34(+) cells for 24 to 36 hours in the presence of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) resulted in a significant increase in MIP-1alpha receptor expression. TNF-alpha induced MIP-1alpha receptor upregulation in a time- and concentration-dependent manner. Our results suggest that inhibitory cytokines produced by the bone marrow microenvironment are likely to be involved in the regulation of MIP-1alpha receptor expression on hematopoietic cells.

Differential response of CD34+ cells isolated from cord blood and bone marrow to MIP-1 alpha and the expression of MIP-1 alpha receptors on these immature cells.

Macrophage inflammatory protein-1 alpha (MIP-1alpha) has been shown to have a role in the control of myeloid stem and progenitor cell proliferation. Recent evidence suggests that MIP-1alpha also has a stimulatory effect on proliferation of mature progenitors as well as an inhibitory effect on immature progenitors in vitro. We have compared the effect of MIP-1alpha on myeloid and erythroid colony formation of CD34+ cells isolated from bone marrow and cord blood. In the presence of MIP-1alpha, bone marrow granulocyte-macrophage-colony forming cells (GM-CFC) were inhibited over a dose range of 15 ng/ml to 500 ng/ml, and GM-CFC from cord blood CD34+ cells were stimulated over the same dose range. MIP-1alpha suppressed BFU-E colonies in both bone marrow and cord blood. Using thymidine suicide assays, the influence of MIP-1alpha on the cycling status of the cells was assessed. A good correlation between the effect of MIP-1alpha on colony formation and cell cycle progression was observed. These results suggest that there is a differential response to MIP-1alpha when bone marrow and cord blood CD34+ cells are compared. Using flow cytometry and a biotinylated human MIP-1alpha/avidin fluorescein conjugate, the expression of MIP-1alpha receptors on CD34+ cells was assessed. The data indicated that there was little quantitative difference in overall expression of receptors (82.9% versus 93%) from bone marrow or cord blood, respectively. However, when Northern blot analysis was used, mRNA for two different MIP-1alpha receptors CCR1 and CCR5 could be detected in bone marrow, but only CCR1 mRNA was seen in cord blood CD34+ samples. Therefore, the expression of different receptor subtypes on CD34+ cells may be responsible for the difference in MIP-1alpha responsiveness observed.

Recombinant human megakaryocyte growth and development factor (MGDF) increases the numbers of megakaryocyte progenitor cells to normal values in long-term bone marrow cultures of patients with AML in first remission.

The megakaryopoietic potential in the bone marrow (BM) of patients in first remission after treatment for acute myelogenous leukaemia (AML) was investigated using long-term bone marrow cultures (LTC) stimulated with megakaryocyte growth and development factor (MGDF). The baseline number of megakaryocyte colony-forming cells (Meg-CFC) was very low. However, there was a 10 to 100-fold increase of Meg-CFC in cultures treated with 10 ng/ml MGDF with mean numbers within the normal range for the first 4 weeks of culture with a 24-fold increase in their cumulative numbers. Similarly, a 12-fold increase in the numbers of megakaryocytes (MKs) was found by CD61 immunostaining. These effects were lost at the dose of 100 ng/ml. In contrast, the cumulative mean numbers of Meg-CFC in the control cultures from normal bone marrow (NBM) were not significantly different from those in cultures treated with 10 or 100 ng/ml MGDF. These results demonstrate that MGDF stimulates megakaryocytopoiesis in patients with AML in first remission, restoring the Meg-CFC compartment to normal values, a result with potential clinical implications for their treatment with autologous transplantation.

Regulation of the proliferative potential of cord blood long-term culture-initiating cells (LTC-IC) by different stromal cell lines: implications for LTC-IC measurement.

BACKGROUND AND OBJECTIVE: Long-term culture-initiating cells (LTC-IC) are the best available approximation to an in vitro assay of stem cells in humans although they still represent a heterogeneous population in terms of proliferative capacity and sensitivity to different growth factors. Human umbilical cord blood (CB) is rich in hemopoietic progenitor cells, as measured by clonogenic assays and contains stem cells capable of reconstituting the marrow after ablation in clinical transplantation. We evaluated the influence of culture conditions on the in vitro behavior of LTC-IC from CB. DESIGN AND METHODS: LTC-IC were evaluated in long-term cultures, comparing two types of murine stromal cell lines: M2-10B4 and M2-10B4 transfected with cDNAs for human G-CSF and IL-3. RESULTS: Two and five fold higher numbers of terminally differentiated cells were produced during nine weeks of culture of CB mononuclear or CD34+ cells respectively, in cultures containing a M2-10B4 IL-3 G-CSF cell line compared to cultures containing the parental cell line. Likewise, a higher number of colony-forming cells (CFC) were detected in the supernatant of cultures with the transfected cell line. In contrast, the number of CFC generated within the stromal layer, after 5 or 9 weeks of culture, was significantly higher in cultures on M2-10B4 cells than those on M2-10B4 IL-3 G-CSF. INTERPRETATION AND CONCLUSIONS: Our results show that the proliferative capacity of CB LTC-IC can be strongly influenced by culture conditions and that the frequency of LTC-IC estimated using these cell lines as stromal support is not identical.

Extensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identification of long-term culture-initiating cells present in two subsets.

CD34+ cord blood cells were isolated with immunomagnetic beads and fractionated by fluorescence-activated cell sorting (FACS) into three subpopulations: CD34+38+DR+, CD34+38-DR+ and CD34+38-DR-, using antibodies specific for these cell surface markers. Cells from each of the three subsets were plated as single cells in serum-free medium supplemented with a combination of growth factor and individual cells were monitored for proliferation and the capacity to form colony-forming cells. Single cells from the CD34+38+DR+ subset showed the lowest expansion capacity, generating up to 1.1 x 10(6) cells at five weeks, while individual cells from both the CD34+38-DR+ and CD34+38-DR- subsets could be expanded up to 1.8 x 10(6) and 9.2 x 10(6) cells, respectively, over a period of six weeks. The different subpopulations also generated colony-forming cells which gave rise to erythroid, myeloid and erythroid/myeloid colonies. CD34+38-DR+ cells generated large numbers of colonies within two weeks in liquid culture, but this rapidly declined. Generation of lineage-committed colony-forming cells was better sustained in the CD34+38-DR- population and continued for up to six weeks in culture. Overall, the generation of colony-forming cells declined with time in culture, although the cell numbers continued to expand. However, when the same populations were plated on irradiated bone marrow stroma, both the CD34+38-DR+ and the CD34+38-DR- cells were capable of producing granulocytemacrophage colony-forming cells (GM-CFCs) for 10 to 12 weeks. As hemopoiesis was sustained for almost three months, it appears that these populations were significantly enriched in long-term culture-initiating cells (LTC-ICs). Although both populations generated GM-CFCs, the CD34+38-DR- cells sustained production of higher numbers of colony-forming cells than the CD34+38-DR+ population. These results demonstrate that cells from cord blood can be efficiently monitored at the single-cell level for proliferation, expansion and colony-forming capacity. Furthermore, at least two populations of LTC-ICs can be distinguished in cord blood CD34+38- cells by the differential expression of the HLA-DR antigen.

Comparison of purity and enrichment of CD34+ cells from bone marrow, umbilical cord and peripheral blood (primed for apheresis) using five separation systems.

Interest in the isolation and characterization of primitive hemopoietic cells in both the clinical and research fields has rapidly increased. In parallel, different purification systems have been developed to isolate these cells. We have compared five different methods for separation of CD34+ cells from human umbilical cord blood, normal bone marrow and apheresis harvests and analyzed purity, recovery, yield and enrichment of colony forming cells (CFC) for each individual system. Our results indicate that the most reliable methods of purification for all samples were fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS) which consistently yielded high purities (> 70%) and enrichment of CFC. In this respect the enrichment of CFC from the MACS was superior to all the other systems including FACS. Similar results (> 70%) for purity were obtained using avidin affinity columns and a biotinylated antibody but neither yield nor CFC enrichment approached the values achieved with MACS. On average CFC enrichment using these affinity columns was greater than that observed for FACS while the purity was comparable. Both CELLector flasks and immunomagnetic beads coated with CD34 antibodies were less effective in our hands in separating purified populations of progenitor cells. Both purity and CFC enrichment of CD34+ cells using these methods were at least 50% lower than obtained with either FACS, MACS or affinity columns.

CD34 cell separation: from basic research to clinical applications.

Advances in the immunological identification of primitive haematopoietic cells have led to the development of various techniques for their characterisation and purification. The expression of the CD34 antigen by the stem cell compartment has been exploited for these purposes. Separation systems capable of isolating CD34+ cells on a large scale are finding use in the clinic. Areas of interest overlap for both researchers and clinicians, and efforts to mobilise, characterise, purify and transplant these cells continue. Different CD34 purification systems are reviewed, focusing on their possible applications in different research fields. Clinical results of CD34+ selection and the possible future applications of this technology in the clinic are also reported.