Cord Blood Stem Cells

Haematopoietic repopulating activity in human cord blood CD133+ quiescent cells


We have demonstrated previously that cord blood CD133+ cells isolated in the G0 phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133+G1 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+G0 cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133+G1 counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133+G0 population. In long-term BM cultures, the CD133+G0 cells generate significantly more progenitors than the CD34+G0 population (P<0.001) throughout the culture period. Furthermore, a comparison of CD133+G0 versus CD133+G1 cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G0 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 G1 counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.


Pluripotent haematopoietic stem cells (HSCs) are a rare subset of cells responsible for the lifelong production of all circulating blood cells. HSCs are defined functionally by their extensive proliferation, differentiation and self-renewal capacity and by their ability to reconstitute the haematopoietic system of an irradiated host. Human umbilical cord blood (CB) is a rich source of HSCs, and this has led to an increase in the use of CB units for allogeneic transplantation. One advantage of CB transplants compared with unrelated BM transplants is the reduced incidence and severity of acute and chronic GVHD.1, 2 However, delayed engraftment is a problem when CB is compared with BMT.3, 4 This is probably because of the number of stem cells present in a unit of CB. A more accurate determination of the CB stem cell content may allow for better prediction of engraftment as well as an indication of the cell dose required for successful transplantation.

Evidence of human haemopoietic stem cell activity can be obtained from transplantation experiments using highly immunodeficient mouse strains, such as non-obese diabetic (NOD)/SCID mice. These animals have reduced residual natural killer cell activity, thereby allowing increased human stem cell engraftment.

Traditionally, the CD34 antigen has been used to select human cells capable of multilineage repopulation, but there is accumulating evidence that not all HSCs express the CD34 antigen. CD34-negative HSCs have been identified and shown to engraft and repopulate irradiated NOD/SCID mice.5, 6, 7 Furthermore, cells with repopulating activity, referred to as the ‘side population’, have been defined on the basis of their ability to efflux the dye Hoechst 33342. This small population exhibits a characteristic pattern of fluorescence in the far red and blue emission channels when analysed by flow cytometry, and are predominantly CD34 negative.8, 9 Another cell-surface glycoprotein, CD133 (formerly known as AC133), is expressed on primitive human progenitor cells and does not share significant homology with any previously described HSC surface antigen.10 CD133 may be a marker for a more primitive cell subset, as CD133-expressing cells generate CD34+ cells in liquid culture.11 Importantly, it has been demonstrated that human AC133+CD7neg cells engraft and produce progeny in the BM of NOD/SCID mice, whereas the AC133negCD7neg subset has no engraftment capability.11 Thus, purification of HSCs on the basis solely of CD34 expression may exclude other repopulating cells.

In previous studies, we have used simultaneous DNA/RNA staining and flow cytometric cell sorting to isolate and characterize CB CD34+ or CD133+ cells in the G0 and G1 phases of the cell cycle. The CD34+G0 cell population has a thousand-fold higher capacity for generating progenitors in vitro than their CD34+G1 counterparts and produce colony-forming cells (CFCs) for up to 20 weeks in culture.12 Subsequently, we demonstrated that the CD133+G0 cells under similar in vitro serum-free conditions contained a high incidence of both CFC and long-term culture (LTC)-initiating cells.13 Thus, at least two of the standard criteria that define stem cells were satisfied by the CD133+G0 subset.

The aim of the study presented here was to define further the phenotype of the CD133+G0 cells and demonstrate the third criterion defining a stem cell in this population, the ability to regenerate the haematopoietic system following transplantation in NOD/SCID mice. Our results indicate that the CD133+G0 subset fulfils the criteria for an HSC population.

Materials and methods

Cell preparation

Human CB cells were obtained from full-term, normal deliveries or elective Caesarean sections, following informed consent. Samples were obtained and used in accordance with the Local Ethics Committee guidelines. The mononuclear cell fraction was separated on Ficoll-Hypaque (Lymphoprep, Life Technologies, Paisely, UK) by density centrifugation and CD133+ or CD34+ cells isolated by magnetic immunoselection (Miltenyi Biotech, Bisley, Germany) as described elsewhere.12, 13

Isolation of G0 and G1 cells

CD133+ or CD34+ cells were incubated in Hoechst buffer (containing Hanks balanced salt solution, 0.1% D-glucose, 20 mM HEPES, 10% FCS) at a concentration of 1–1.5 × 106 cells/ml. Hoechst 33342 (Sigma-Aldrich, Gillingham, Dorset, UK) was added to give a final concentration of 1 μg/ml and the cells were incubated for 45 min at 37 °C. Pyronin Y (Sigma-Aldrich) was then added to the cell suspension to a final concentration of 0.5 μg/ml and incubation was continued for a further 45 min. The cells were then washed in Hoechst buffer and labelled with CD34-FITC antibody, Clone 8G12 (BD Biosciences, San Jose, CA, USA) or CD133/2-Biotin (Miltenyi Biotech) followed by streptavidin-FITC secondary antibody (BD Biosciences) at 4 °C for 20 min. After washing, the cells were sorted on either a FACSVantage (BD Biosciences) or MoFlo (Dako, Glastrup, Denmark) flow cytometer equipped with argon and UV lasers.

LTC of CD34 or CD133 populations of G0 or G1 cells on stromal layers

M210 B4 stromal cells were cultured in T25 culture flasks and the prepared stromal layers were irradiated with 80 Gy. LTCs were initiated by adding 5 × 103 freshly sorted cells from populations of CD34+ or CD133+ cells in G0 or G1 phase to the stromal layers. The cultures were maintained in medium consisting of Iscoves's modified Dulbecco's medium (Gibco, Invitrogen, Paisely, UK), 10% FCS (v/v), 10% horse serum (v/v) (Stem Cell Technologies, Vancouver, Canada, SARL, UK) and 5 × 10−7 M hydrocortisone-21-succinate (Sigma-Aldrich) for 8 weeks with a weekly change of half of the medium. At weeks 5 and 8, the number of progenitor cells in the adherent layer was determined by removing all the culture supernatant, detaching the adherent stromal layer with trypsin and assaying the detached cells for the presence of haematopoietic progenitors.

Progenitor cell assays

To assay haematopoietic progenitors, 2 × 103 harvested cells were resuspended in 100 μl of Iscoves's modified Dulbecco's medium and added to 900 μl of MethoCult H4230 (Stem Cell Technologies) containing 5 μg/ml SCF, 1 μg/ml IL-3, 5 μg/ml GM-CSF and 2.5 U/ml EPO (R&D Systems, Oxford, UK). Cultures were plated in triplicate. Plates were incubated at 37 °C, 5% CO2, for 14 days and colonies were scored according to standard criteria.

Transplantation and analysis of human engraftment

All animal experiments were performed in compliance with UK Home Office and institutional guidelines. NOD/SCID and NOD/SCID β2-microglobulin (β2M)-null mice were originally obtained from Dr Leonard Schultz (Jackson Laboratory, Bar Harbour, ME, USA) and bred at Charles Rivers Laboratories, UK. They were kept in micro-isolators and fed with sterile food and acidified water. Mice aged 8–12 weeks were irradiated at 375 Gy (137Cs source) up to 24 h before intravenous injection of cells. Mice were killed 8–10 weeks after transplantation. The femurs, tibias and pelvis were dissected and flushed with PBS to obtain the BM cells. RBCs were lysed by treating with 0.8% ammonium chloride. Cells were stained with human-specific FITC-conjugated anti-CD19, phycoerythrin-conjugated anti-CD33 and phycoerythrin-Cy5-conjugated anti-CD45 antibodies (BD Biosciences). Dead cells and debris were excluded through diamidino-phenylindole staining and analysed by flow cytometry. More than 100 000 diamidino-phenylindole-negative events were collected. Multilineage engraftment was defined as two separate populations of CD45+CD33+ and CD45+CD19+, cells with the appropriate forward- and side-scatter profiles.

Phenotypic analysis of CD133+ G0 and G1 cells

Cord blood CD133+ cells were isolated as described previously and sorted into three subsets according to their DNA and RNA contents (G0, early G1 and late G1). The cells were stained for the presence of aldehyde dehydrogenase (ALDH) using the Aldefluor kit (Aldagen, Durham, NC, USA) according to the manufacturer's instructions. Briefly, cells were resuspended in Aldefluor assay buffer at 1 × 106 cells/ml with 5 μl/ml Aldeflour substrate with or without 5 μl/ml diethylaminobenzaldehyde, an inhibitor of ALDH activity used to set baseline fluorescence.14 The cells were incubated at 37 °C for 30 min, then washed and resuspended in assay buffer and stained with anti-human CD133/2-APC (Miltenyi Biotech), CD34-PerCP Cy5.5 and CD38-phycoerythrin-Cy7 (BD Biosciences), keeping the cells on ice at all times. Analysis of the wells was performed on an LSRII flow cytometer (BD Biosciences). Gates were set according to the appropriate isotype and diethylaminobenzaldehyde controls.

Statistical analysis

The different cell subsets were compared by Student's t-test or Mann–Whitney U-test, whichever was appropriate using a statistical software package (SPSS version 15.0). A P-value of 0.05 was considered to be statistically significant.


FACS of CD133+G0 and CD133+G1 cell subsets

CD133+ cells were enriched from CB using immunomagnetic beads (Figure 1a), and the G0 and G1 subsets were then purified by FACS.13 Cell cycle analysis with Hoechst 33342 dye revealed that all CD133+ cells were in the G0/G1 phase (Figure 1b). The sorting gates for G0 and G1 are illustrated in Figure 1c and were set according to low and high expression of Pyronin Y. The G0 gate contains cells with a diploid DNA content and low RNA content, whereas cells with higher RNA content are gated as G1. The G0:G1 ratio of the CD133+ cells was approximately 2:1. It should be noted that this ratio is reversed among CD34+ cells selected from umbilical CB in G0 or G1 phase of the cell cycle, where the G0:G1 ratio is 1:2 (Wilpshaar et al.15). The number of mononuclear cells used in the selection procedure and the number of CD133+ cells recovered from CB samples are shown in Table 1. In 21 samples, an average of 1.4 million CD133+ cells were recovered from 117 million mononuclear cells.

Figure 1

Staining of cord blood CD133+ cells and selection of G0 and G1 cells. (a) Gating of CD133+ cells following selection with CD133-conjugated immunomagnetic beads. (b) Hoechst staining showing all CD133+ cells in G0/G1 phase. (c) CD133+ cells stained with Hoechst/pyronin Y; G0 and G1 sorting gates are as shown.

Table 1 Summary of cell content and recovery of CD133+ cells from CB samples

Cells that initiate long-term culture reside among the G0 subset

We compared four cell populations, CD133+G0, CD133+G1, CD34+G0 and CD34+G1, all isolated from a single CB sample, for their expansion potential and production of progenitor cells in long-term culture over eight weeks (Figure 2). Both GM-CFC production and the number of non-adherent cells were evaluated weekly and are presented as numbers per flask. From the outset, the number of GM-CFC produced by CD133+G0 or CD34+G0 cells in LTC was higher than those produced by their counterparts in G1 phase of the cycle. When GM-CFC production from the CD133+G0 and CD34+G0 cultures was compared, there was a significantly higher generation of progenitors from the CD133+G0 cells (P<0.001). Similarly, the expansion of non-adherent cells in the CD133+G0 cultures was also significantly higher than those of the CD34+G0 cultures (P<0.0002). In contrast, there was little difference in expansion between the CD34+G1 and CD133+G1 cells and no difference in the quantities of progenitor cells generated during the culture period (P<0.11 and 0.41, respectively). The cells that generate progenitors in LTC reside and proliferate in the adherent stroma; we therefore assayed the adherent stroma from the four different cultures for progenitor cells at weeks 5 and 8. At both time points, there were much greater numbers of progenitors in the adherent layer of the CD133+G0 cultures compared with the CD34+G0 cultures (Figure 3). Progenitor cells were not detectable in the adherent stroma from the CD133+G1 or CD34+G1 cultures at either time point (data not shown). These experiments indicated that long-term growth in vitro was primarily associated with cells in the quiescent phase of the cell cycle and that CD133+ cells had much greater proliferative potential than CD34+ cells. Importantly, these results were obtained from individual CB samples, so that even though there may have been sample-to-sample variation, the ranking of the most prolific cell populations was consistent.

Figure 2

Long-term culture of CD34+ or CD133+ cells in G0 or G1, isolated from a single cord blood (CB) sample. The four cell populations were purified by FACS, plated on irradiated M210B4 stroma and cultured for 7–8 weeks as described in Materials and methods. Cultures were initiated with 5000 cells from CD133+G0, CD133+G1, CD34+G0 or CD34+G1 populations from one individual CB sample. On the left, graphs illustrating generation of progenitor cells (GM-CFC) and on the right the corresponding experiment showing expansion of the non-adherent nucleated cells in the culture supernatant as a function of time are shown. Three separate experiments are shown.

Figure 3

Progenitor cells in adherent layer of long-term cultures (LTCs). Numbers of GM-CFC at weeks 5 and 8 in the stromal layer of LTCs initiated with the CD133+G0 or CD34+G0 cells. No progenitors were obtained from cultures initiated with CD133+G1 or CD34+ G1 cells (data not shown).

NOD/SCID repopulating cells reside in the CD133+G0 population

At present, the definitive test for human HSC activity is the ability to repopulate the haemopoietic system of the NOD/SCID mouse. Purified CD133+G0 and CD133+G1 cells were transplanted into irradiated NOD/SCID mice and human haematopoietic engraftment was monitored 8–10 weeks after transplantation in five separate experiments. The results from a total of 19 animals are summarized in Figure 4a. At doses of 20 000, 10 000 or 5000 CD133+G0 cells, engraftment of human cells (defined as greater than 0.05% of total live cells) could be detected in the BM of mice receiving the transplant. In contrast, transplantation of equivalent doses of CD133+G1 cells resulted in no detectable human haematopoietic cells. About half of the animals injected with CD133+G1 cells failed to survive over the 8-week period of the experiments (data not shown). Nevertheless, the data indicated that at a dose of 10 000 cells per animal, there was a significant difference in the engraftment levels between CD133+G0 and CD133+G1 cells (P<0.01). Engraftment levels can be increased using NOD/SCID β2M-null mice,16, 17 and in line with this, we observed higher levels of engraftment when we injected 10 000 CD133+G0 cells into three NOD/SCIDβ2M-null animals (Figure 4b), with up to 31% human CD45+ cells detected at week 8. None of the animals injected with a similar number of CD133+G1 cells showed engraftment above the threshold level. These results reveal that repopulating activity resides in the CB CD133+G0 fraction, indicating that human HSCs reside in this population.

Figure 4

Haematopoietic engraftment of CD133+G0 and CD133+G1 cells in non-obese diabetic (NOD)/SCID mice. (a) Summary of engraftment of NOD/SCID mice transplanted with 20 000, 10 000 or 5000 CD133+G0 or CD133+G1 cells in three experiments. The graph shows the percentage of human CD45+ cells in the NOD/SCID BM 8–10 weeks post-transplantation. Each data point represents a single mouse and the dotted line shows the engraftment threshold (set at 0.05%). (b) Results from six NOD/SCIDβ2M-null mice transplanted with either 10 000 CD133+G0 or CD133+G1 cells and assessed at 8 weeks post-transplantation for CD45+ cells in the BM.

CD133+G0 cells exhibit multilineage engraftment

The BM from animals engrafted with the CD133+G0 cells was then analysed for the presence of lymphoid and myeloid lineage cell surface markers (CD19 and CD33, respectively). Analysis showed the presence of CD45+/CD19+ and CD45+/CD33+ cells in the animals repopulated with CD133+G0 cells, but not in those animals receiving CD133+G1 cells (Figures 5a and b). Engraftment of CD45+/CD19 ranged from 0.22 to 10.3%, whereas CD45+/CD33+ engraftment was 0.3–7.6%. Mature T-cells cannot be detected in the BM of the NOD/SCID mouse.18 We also examined the BM from transplanted animals for the expression of the human CD34 antigen as an indicator of the maintenance of primitive cells. Our results indicate that a significant proportion of human cells (0.05–2.6%) within the marrow of the NOD/SCID mice expressed CD34 (Figure 4c), consistent with the generation of CD34+ cells from the transplanted CD133+ cells. This is also in line with other reports showing that CD34+ cells are generated from CD133+ cells in vitro.11

Figure 5

Multi-lineage engraftment of CD133+G0 cells in non-obese diabetic (NOD)/SCIDβ2M-null BM. FACS analysis of BM cells from an individual NOD/SCIDβ2M-null mouse transplanted with 10 000 CD133+G0 or 10 000 CD133+G1 cells. Analyses were performed at 9 weeks after transplantation. (a) Proportion of cells expressing human CD45 in the CD133+G0- or CD133+G1-transplanted animals. (b) Expression of myeloid (CD33+) and B-lymphoid (CD19+) markers in the CD45+ population from the CD133+G0 transplanted animal (no CD45+ cells were present in the CD133+G1-transplanted animal). (c) Human CD133+ and CD34+ cells were found in the BM of mice engrafted with CD133+G0 cells.

Phenotype of CD133+G0 cells

Cells expressing CD34 but lacking the CD38 antigen have long been considered to be an enriched HSC population.19, 20 More recently, ALDH activity has been used to define distinct CD34+ stem and progenitor cell compartments in human umbilical CB.14 Primitive human haemopoietic cells contain higher levels of ALDH than their terminally differentiating progeny. It has been confirmed that CFCs have less ALDH activity than LTC-initiating cells and that isolation of ALDH cells enriches all types of repopulating cells and can be a useful predictive as well as preparative procedure.21 We therefore analysed the CD133+G0 population for the expression of CD34, CD38 and ALDH expression. As shown in Figure 6, the CD133+G0 population is enriched in CD34+CD38neg cells and have high ALDH activity when compared with the early and late G1 populations. This phenotype becomes less prominent as the cells progress through G1. Thus, the markers currently associated with human HSC activity are indeed present within the CD133+G0 subset.

Figure 6

Phenotype of CD133+G0 and CD133+G1 cell-cycle phase. (a) Cord blood CD133+ cells were sorted according to the phase of cell cycle; G0, early G1 and late G1. Cells were subsequently stained with (b) CD34 and CD38 antibodies and (c) for aldehyde dehydrogenase activity (ALDH). The gate for ALDH activity was set using the diethylaminobenzaldehyde inhibitor. FACS analysis shown is representative of three individual samples.


A major goal of clinical transplantation is to identify primitive HSCs with high repopulating ability. At present, the parameter that correlates best with the outcome of a CB transplant is colony number and not CD34 frequency. Although there are reports indicating that CD133-selected cells might be superior to CD34+-selected cells for SCT, CD133 frequencies have not been assessed routinely in transplantation protocols.11, 22

We have demonstrated previously that differences exist in the proliferative potential of CB cells isolated in the G0 and G1 phases of the cell cycle. The CD133+G0 cells (formerly denoted as AC133+G0) have a higher incidence of LTC-initiating cells and produce significantly more nucleated cells and CFCs in serum-free, stroma cell-free conditions compared with CD34+G0 cells.12, 13 The extended period over which G0 cells are able to maintain production of progenitors and the magnitude of the production suggests that HSCs may be undergoing self-renewal in the serum-free cultures. Thus, both the CD133+G0 cell subset and the culture conditions are well suited to ex vivo expansion protocols, if expanded cells are to be used in the clinical setting. We now show that when CD133+G0 and CD34+G0 are isolated from the same CB sample and used to initiate stroma-supported LTCs, CD133+G0 cells are significantly superior to CD34+G0 cells in their ability to generate progenitor cells (P<0.001). These results argue that an enriched stem cell population is present among the CD133+G0 cells.

During steady-state haemopoiesis, the HSC population is relatively quiescent and long-term engraftment capacity of transplanted HSC depends critically on maintenance of the quiescent state.23, 24, 25 HSCs in adult mobilized peripheral blood capable of engrafting NOD/SCID mice are found predominantly in the G0 phase of the cell cycle.26 In CB, both CD34+G0 and CD34+G1 cells could repopulate NOD/SCID mice, but the level of engraftment obtained in the mice transplanted with CD34+G0 cells was greater than that found in those receiving CD34+G1 cells.15 However, HSC activity is not confined to the CD34+ population, and HSC activity has been found within the CD34neg population.27 There is discordant expression of CD133 and CD34 in human haematopoietic tissue,11, 28, 29, 30 so we analysed the HSC activity within CD133+G0 and G1 cells and now show that the NOD/SCID repopulating activity resides within the CD133+G0 population. It is difficult to directly compare the repopulating activity observed in our study with that found for other subsets in previous reports because such analyses are complicated by different isolation procedures that can affect sample quality (pooled versus unpooled samples), variability within NOD/SCID mice, inclusion of accessory cells and the irradiation dose and source. Nevertheless, the level of engraftment that we observe with CD133+G0 cells alone is not dissimilar to that obtained by others using target cells in combination with accessory cells.22, 31 In future studies, it will be important to directly compare HSC activity in CD34+G0 and CD133+G0 cells and to perform secondary/tertiary transplants to assay long-term repopulating cells. Whether CD133+G0 cells have intrinsically higher HSC activity or whether they have superior BM homing efficiency compared with their G1 counterparts also remains to be elucidated. In either case, this would be advantageous for clinical transplantation.

The CD133+G0 fraction contains a large proportion of CD34+38 and ALDH+ cells, markers previously shown to be associated with HSC activity, and thus indicating that the CD133+G0 population is a valid source of functional stem cells.14, 19, 20, 32 The primitive quiescent cells do not take up the intercalating DNA dye in this selection procedure, so the potential to cause DNA damage is reduced. Hence, our purification strategy for CB cells may be compatible with clinical use.

It is possible that CD133+ cells, or CD133+ cells in G0, may be a better predictor than colony numbers for the outcome of the transplant. If this could be confirmed, it would be a considerable asset in determining the suitability of CB units for transplantation.

In summary, we have confirmed that functional HSC activity resides in the CD133+G0 subset, and that this population has in vivo repopulating activity and contains a high proportion of CD34+CD38neg cells as well as cells with high ALDH activity. Defining the phenotype of human HSCs using these parameters has enhanced basic stem cell research33 and has potential for future clinical applications. There are many CB transplantation-related issues that are still awaiting investigation, and some of these could be addressed by further study of the frequency and functional behaviour of CD133+G0 cells.


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This work was supported by Yorkshire Cancer Research and Cancer Research UK, SJH European Commission Consert Grant 005242 and MPB EU Grant: EURO-POLICY-PID: Ref: SP23-CT-2005-006411. We are grateful to Liz Straczynski and Adam Davison for assistance with flow cytometry, our colleagues for cooperation with cord blood collection and Adrian Thrasher for support. We would like to thank Dr L Miall and staff of the Antenatal Unit at St James's University Hospital for their assistance with cord blood collection.

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Correspondence to E A de Wynter.

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Boxall, S., Cook, G., Pearce, D. et al. Haematopoietic repopulating activity in human cord blood CD133+ quiescent cells. Bone Marrow Transplant 43, 627–635 (2009).

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  • haematopoietic stem cells
  • cord blood
  • NOD/SCID mice
  • CD133
  • aldehyde dehydrogenase activity

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