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TISSUE-SPECIFIC STEM CELLS

In Vitro Expanded Cells Contributing to Rapid Severe Combined Immunodeficient Repopulation Activity Are CD34⁺38^–33⁺90⁺45RA^–

^aDepartment of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Ghent University Hospital, Ghent, Belgium;
^bDienst voor het Bloed, Rode Kruis-Vlaanderen, Ghent, Belgium

Key Words. Hematopoiesis • Stem cell • Stem cell culture • Nonobese diabetic/severe combined immunodeficient mice

Correspondence: Bart Vandekerckhove, M.D., Ph.D., Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, 4 Blok A, De Pintelaan 185, B-9000 Ghent, Belgium. Telephone: 32(0)9-240-60-65; Fax: 32(0)9-240-36-59; e-mail: Bart.Vandekerckhove{at}Ugent.be

Received on April 25, 2006; accepted for publication on September 1, 2006.

First published online in STEM CELLS EXPRESS  September 14, 2006.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Expansion of hematopoietic stem cells could be used clinically to shorten the prolonged aplastic phase after umbilical cord blood (UCB) transplantation. In this report, we investigated rapid severe combined immunodeficient (SCID) repopulating activity (rSRA) 2 weeks after transplantation of CD34⁺ UCB cells cultured with serum on MS5 stromal cells and in serum- and stroma-free cultures. Various subpopulations obtained after culture were studied for rSRA. CD34⁺ expansion cultures resulted in vast expansion of CD45⁺ and CD34⁺ cells. Independent of the culture method, only the CD34⁺33⁺38^– fraction of the cultured cells contained rSRA. Subsequently, we subfractionated the CD34⁺38^– fraction using stem cell markers CD45RA and CD90. In vitro differentiation cultures showed CD34⁺ expansion in both CD45RA^– and CD90⁺ cultures, whereas little increase in CD34⁺ cells was observed in both CD45RA⁺ and CD90^– cultures. By four-color flow cytometry, we could demonstrate that CD34⁺38^–45RA^– and CD34⁺38^–90⁺ cell populations were largely overlapping. Both populations were able to reconstitute SCID/nonobese diabetic mice at 2 weeks, indicating that these cells contained rSRA activity. In contrast, CD34⁺38^–45RA⁺ or CD34⁺38^–90^– cells contributed only marginally to rSRA. Similar results were obtained when cells were injected intrafemorally, suggesting that the lack of reconstitution was not due to homing defects. In conclusion, we show that after in vitro expansion, rSRA is mediated by CD34⁺38^–90⁺45RA^– cells. All other cell fractions have limited reconstitutive potential, mainly because the cells have lost stem cell activity rather than because of homing defects. These findings can be used clinically to assess the rSRA of cultured stem cells.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
During the past decade, umbilical cord blood (UCB) has been established as an alternative source of hematopoietic stem cells (HSC) for transplantation. Cord blood transplantation has several advantages over bone marrow (BM) or peripheral blood stem cell (PBSC) transplantation, such as a lower incidence of graft-versus-host disease, which allows partial matching between donor and recipient. However, there are major disadvantages. First of all, 1 UCB unit contains a limited number of HSC. Because the number of CD34⁺ cells and total nucleated cells transplanted has a significant effect on engraftment and survival of the patient, the use of UCB for adult transplantation is limited. Moreover, UCB grafts have a mean time to neutrophil engraftment of 26 days, which is much longer than for BM or PBSC grafts [1–3].

In vitro expansion of stem cells with rapid severe combined immunodeficient (SCID) repopulating activity (rSRA), even in the absence of expansion of stem cells with self-renewal and long-term SCID repopulation activity (LT-SRA), may counter the disadvantages of UCB as a stem cell source. The term rapid SCID repopulating cells (rSRC) has been introduced by Mazurier et al. [4] to describe cells that rapidly, typically within 2–3 weeks, repopulate nonobese diabetic (NOD)/SCID mice. The contribution of multipotent long-term hematopoietic stem cells (LT-HSC) and short-term HSC (ST-HSC) or more committed precursors to this activity is not well studied. We developed a model for the study of rapid repopulating cells and subsequently characterized the cells with this activity. We found that NOD/SCID mice, if pretreated with mouse CD122 antibody, are an excellent model for the study of rSRA [5]. rSRC as well as LT-SRC engraft in these mice. This was confirmed by other groups [6, 7]. In this model, we and others could show that CD34⁺38⁺ and not CD34⁺38^– cells were responsible for the rapid outgrowth of human cells in these mice [4, 8, 9]. After 7 days, already substantial repopulation of the BM could be observed, and after 4 weeks, human CD34⁺ cells were no longer detectable, indicating a rapid and short term repopulating activity of these CD34⁺CD38⁺ cells. On the other hand, CD34⁺38^– cells were not detectable in the BM until 4 weeks after transplantation and started to increase after that time. Different kinetics of CD34⁺ cells, depending on the expression of CD38, were also demonstrated by other research groups, although minor differences were noted, probably attributable to different sorting gates for CD38 [4, 8, 9].

Although various reports show no or only limited increase in SRC after ex vivo expansion [10–16], it is widely assumed that in vitro-expanded cells are a rich source of more committed cells, possibly including rSRA [17]. Indeed, it is known that after in vitro culture, CD34⁺ cells and colony-forming unit and burst-forming unit-erythroid (BFU-E) numbers increase dramatically. Therefore, some assume that mainly the more mature precursors, which may include ST-HSC and lineage restricted precursors, are increased [15, 18]. However, clinical data demonstrate that transplantation of in vitro-expanded CD34⁺ cells is tolerated well but does not dramatically shorten the time to neutrophil engraftment [17, 19, 20]. It is difficult to address whether this is due to a reduction of cells with rSRA, especially because the cells responsible for rSRA after in vitro culture are not well characterized.

Little is known about the phenotype or engraftment characteristics of in vitro-cultured multipotent and lineage-committed precursors that may have rSRA. It has been reported that the phenotype of fresh stem cells is no longer valid after culture, as an increase in CD34⁺38^– cells does not correlate with SRC [11]. Moreover, more than 90% of generated CD38^– cells express the early myeloid markers CD13 and CD33 [11]. CD90, on the other hand, was reported to correlate well with stem cell activity both in fresh and in cultured cells [15, 21, 22].

Most studies concentrate on one culture condition, thus evoking the concept that the results obtained can be restricted to and dependent on this culture condition. We used two widely different culture conditions for our studies. One condition consisted of serum-free medium and high cytokine concentrations without stromal cells. These culture conditions have been studied extensively, especially in a (pre)clinical setting [14, 23]. An important increase after 14 days of culture was reported in CD34 cell numbers, colony-forming unit-granulocyte macrophage, and long-term culture-initiating cells (LTC-IC) [18]. We choose to add a serum-containing, stroma-containing condition on one hand because several reports demonstrated a beneficial effect of stromal cells on self-renewal of proliferating stem cells [24–28] and on the other hand to demonstrate that the phenotypic analysis was not exclusive for one culture condition. Here, we demonstrate that rSRA is confined to a small subset of CD34⁺ cells with a CD34⁺CD38^–CD33⁺CD90⁺CD45RA^– phenotype.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Monoclonal Antibodies
Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, and allophycocyanin (APC)-conjugated monoclonal antibodies (mAbs) were purchased from BD Biosciences (Erembodegem, Belgium, http://www.bdbiosciences.com) except for CD7PE, CD15PE, and CD56APC, which were obtained from Immunotech (Beckman Coulter, Marseille, France, http://www.immunotech.com). Anti-murine FcRII/III mAb (2.4G2; kind gift of Dr. J. Unkeless, Mount Sinai School of Medicine, New York) was used to block nonspecific Fc receptor binding.

Mice
Nod/LtSZ-scid/scid (NOD/SCID) breeding pairs, originally purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org), were bred in isolator cages and fed sterilized food. Animals were treated during the course of the experiment according to the guidelines of the Laboratory Animal Ethical Commission of Ghent University.

Cell Sources
UCB was obtained from the cord blood bank of the Red Cross. Mononuclear cells were isolated within 24 hours after collection by a density gradient (Lymphoprep; AXIS-Shield PoC AS, Oslo, http://www.axis-shield.com). CD34⁺ cells were isolated using anti-CD34-tagged super-paramagnetic microbeads according to the protocol of the company (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). Purity of the cells after two passages through the column was always measured by flow cytometry. Unless used fresh, cells were frozen in heat-inactivated fetal calf serum (Perbio; Hyclone, Erenbodegem-Aalst, Belgium, http://www.hyclone.com) supplemented with 10% dimethyl sulfoxide (Serva, Heidelberg, Germany, http://www.serva.de) and stored in liquid nitrogen until use.

In Vitro Differentiation Assay
Sorted subpopulations of cultured cells were incubated (500– 1,000 cells per well) in 24-well plates precoated with confluent murine marrow-derived MS5 cells (kindly provided by L. Coulombel, Institut Gustave Roussy, Villejuif, France) in Iscove's modified Dulbecco's medium (Invitrogen, Merelbeke, Belgium, http://www.invitrogen.com) supplemented with 5% human serum and 5% fetal calf serum (FCS). For assessment of CD34 maintenance and myeloid differentiation, the following mixture of six human recombinant cytokines was used: 50 ng/ml stem cell factor (SCF), 50 ng/ml Flt3L, 20 ng/ml interleukin (IL)-7, 10 ng/ml IL-15, 5 ng/ml IL-2, and 20 ng/ml thrombopoietin (TPO) (mixture 6). For assessment of B lymphoid differentiation, 50 ng/ml SCF and 20 ng/ml IL-7 were used (mixture 2). For the assessment of natural killer (NK) cells, 50 ng/ml SCF, 5 ng/ml IL-2, and 10 ng/ml IL-15 (mixture 3) were used (all reagents from R&D Systems, Abingdon, U.K., http://www.rndsystems.com; and Peprotech, Rocky Hill, NJ, http://www.peprotech.com; except for SCF, which was a generous gift from Amgen, Brussels, Belgium, http://www.amgen.com).

Quantitative analysis was performed by flow cytometry after 2–3 weeks of culture with Flow-Count Fluorospheres, as described below. The phenotype was assessed by flow cytometry after labeling with the following mAbs: CD34APC, CD19PE, CD10PE, CD14PE, CD15PE, CD56APC, CD3PE, and CD45FITC. CD19 and CD10 were used for B cell development, CD14 and CD15 for myelomonocytic development, and CD56 and CD3 for NK development.

In Vitro Expansion
Fresh or frozen and thawed CD34⁺ cells were cultured for 6 days in serum-free medium (Stemspan SFEM; Stem Cell Technologies, Meylan, France, http://www.stemcell.com) supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml) and L-glutamine (2 mM) purchased from Gibco (Grand Island, NY, http://www.invitrogen.com) and the following growth factors: 100 ng/ml SCF, 100 ng/ml Flt3L, and 20 ng/ml TPO. A total of 10⁴ cells per well were seeded in 96-well flat-bottomed tissue culture plates (BD Biosciences). These cultures were designated serum-free (SF) cultures. For MS5 cultures, 3 to 3.5 x 10⁵ cells were incubated per a 75-cm² flask precoated with confluent murine marrow-derived MS5 cells in similar medium except for the addition of 10% heat-inactivated FCS and lower cytokine concentrations (SCF, Flt3L, and TPO at 10 ng/ml). Sixteen milliliters of medium was added per flask. At the end of the cultures, cells were harvested, washed once, and counted by microscope. Cells were injected after culture or were first sorted for the desired phenotype. CD34⁺ cells cultured on stroma were depleted of MS5 cells before injection or cell sorting as follows: cells were labeled with human CD45-biotin mAb (BD Biosciences) and subsequently purified using BD Imag Streptavidin Particles Plus-DM, following the protocol of the supplier. Depletion of stromal cells was checked by flow cytometric analysis and light microscopy.

NOD/SCID Repopulation Assay
Mice that averaged 8 weeks old were given a sublethal dose of whole-body irradiation (300 cGy over a 1-minute period) using an SL75–5 Elekta linear accelerator (Elekta, Zaventem, Belgium, http://www.elekta.com). The mice were injected intraperitoneally with 200 µg of mouse CD122 (purified supernatants of the hybridoma cell line TMß1, kindly provided by Dr T. Tanaka, Tokyo, Japan) to eliminate remaining NK cell (and monocytic) activity [7]. One day later, the human cells were injected intravenously (IV), in the tail vein. Two weeks (rSRA) after injection, the mice were sacrificed, and BM of both femora was harvested. Cell suspensions were filtered through a 70-µm cell strainer (Falcon; BD Biosciences). Red blood cells were lysed using hypotonic lysing buffer. Cells were counted under the microscope and subsequently labeled with the desired mAb. CD45FITC or CD45PE was included in all flow cytometric analyses to measure the percentage of human leukocytes, as shown in Figure 1A. Percentage chimerism (percentage of human CD45⁺ cells) was measured. Differentiation across the various hematopoietic lineages was assessed using CD10 for early B cell development, CD14 and CD15 for myelomonocytic development, and CD56 and CD3 for NK development. We used the early lymphoid marker CD10 as pro-B cell marker since this marker allowed a better distinction between positive and negative populations than CD19 early after transplantation. Total cell numbers per mouse were calculated using the following formula: the absolute number counted in both femora multiplied by 6.25, as both femora represent 16% of the total mouse BM [29]. For intrafemoral (IF) injections, sublethally irradiated mice (injected with CD122) were anesthetized, and a small incision was made over the kneecap. Using a Hamilton 1705 TLL 50-µl syringe (Filterservice, Oudenaarde, Belgium, http://www.filterservice.be) with a 28-gauge needle, access to the femoral BM cavity was gained by perforating the distal femoral epiphysis. A total of 15 µl of cell suspension was injected. Analysis was performed 2 weeks after transplantation. Cell suspensions obtained from injected and not injected femora were analyzed separately by flow cytometry.

View larger version (31K): [in this window] [in a new window]   Figure 1. Comparison of rapid severe combined immunodeficient repopulating activity of fresh and cultured CD34+ cells. (A): Percentage of human CD45+ cells (R2) and CD45+CD34+ cells (R3) determined on a life gate (R1). (B): Percentage of chimerism in the bone marrow (BM) 2 weeks after injection of the indicated cells. (C): Phenotypic analysis of the BM. The cells were stained with human CD45 and CD34 to detect progenitor cells, a mixture of CD14 and CD15 to detect myelomonocytic cells, CD10 to detect B-lineage committed cells, and CD56 and CD3 to detect natural killer cells. The figure shown is a representative experiment of four experiments performed with MS5-cultured cells. Four experiments with serum-free cultured cells gave similar results; mean ± SD is presented. Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; PI, propidium iodide.

Statistical Analysis
Statistical analysis was performed using the Mann-Whitney test for nonparametric comparisons. All p values given are two-sided. Results are expressed as the mean ± SD. Statistical significance was assumed for p < .05.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Expanded UCB CD34⁺ Cells Display rSRA
CD34⁺ cells were isolated with a mean purity of 93.4% ± 4.7% and expanded either on MS5 stromal cells with low concentrations of SCF, TPO, and Flt3L (MS5 cultured) or in serum-free medium with high growth factor concentrations (SF cultured) as described in Materials and Methods. After 6 days of culture, CD34⁺ cells made up 67.1% and 32.0% of the cells and had expanded 27.4- and 9.8-fold under MS5 and SF conditions, respectively. After 12–14 days' expansion, CD34⁺ cell numbers were increased 202.0- and 35.8-fold, respectively (data not shown). To test rSRA, cells were IV injected in irradiated NOD/SCID mice pretreated with CD122. We determined percentage chimerism as shown in Figure 1A by measuring the percentage of human CD45-positive cells on the total viable cell fraction. In an experiment using day 6 MS5 cultured cells shown in Figure 1B, percentage chimerism was 25.6% ± 2.5% for 10⁵ fresh cells and 10.9% ± 2.5% for the progeny of 10⁵ cells after culture. The CD34^– progeny, injected as a control, did not repopulate (Fig. 1B). The difference in chimerism between both fresh and cultured CD34⁺ cells (p < .05) and between both CD34⁺ fractions and CD34^– (p < .001) was significant. NK and myeloid differentiation did not differ significantly, but cultured cells gave rise to a higher percentage of CD10⁺ B lineage cells than fresh cells (Fig. 1C). The percentage of CD34⁺ cells was similar for both conditions (Fig. 1C). Although there was considerable variability, chimerism was always lower for cultured cells compared with fresh cells, independent of the method (MS5- or SF-cultured) or duration (6–14 days) of culture.

Phenotypic Analysis of Expanded Cells
To enable us to determine which cells had rSRA after expansion, cultured cells were analyzed by flow cytometry using various stem cell markers (CD45RA, CD90) and early (CD7, CD10, CD33) and late (CD14, CD15, CD19, CD56) lineage markers. MS5 cultured CD34⁺ cells retained a normal distribution of CD38, as shown in Figure 2. In serum-free medium, CD34⁺CD38⁺ cells virtually disappeared. Although CD45RA expression was generally brighter on CD38⁺ cells, CD38⁺ (in MS5 cultures) and CD38^– fractions contained CD45RA-negative, -low, and -high cells. CD90⁺ cells were detectable in the CD38^– fraction but virtually absent in the CD38⁺ fraction. Disregarding CD38 staining pattern, CD45RA and CD90 stained very similarly, independent of the culture conditions, as 90 and 9%, respectively, of the CD34⁺ cells were positive for these markers.

View larger version (32K): [in this window] [in a new window]   Figure 2. Phenotype of cultured cells. Data are representative of three independent experiments. MS5 and SF cultured CD34+ cells were analyzed by flow cytometry for the indicated markers. First dot plot of both rows represents CD34 gating; cells shown are forward scatter-propidium iodide-gated. Dot plots representing CD45RA, CD90, CD7, and CD33 expression are also gated on CD34+ cells. Quadrants were set according to IgG isotype controls. Abbreviations: APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; SF, serum-free.

Functional Analysis of the Various Cell Populations
Based on the phenotypic analysis described above, three cell populations from MS5 cultured CD34⁺ cells were sorted by fluorescence-activated cell sorting and functionally analyzed: CD34⁺38⁺33^dim, CD34⁺38⁺33⁺, and CD34⁺38^–33⁺. These populations were studied for the ability to maintain or expand CD34⁺ cells and/or to differentiate toward B, NK, and myeloid cells in vitro, as described in Materials and Methods. Analysis after 2 weeks of culture showed that only CD34⁺38^–33⁺ cells could expand CD34⁺ cell numbers, in line with the more immature phenotype of these cells. Not only the percentage CD34⁺ cells was higher in this subfraction (Fig. 3A), but also the absolute numbers: CD45⁺ expansion in the CD34⁺38^– cultures was on average 14 times higher than in the CD34⁺38⁺33⁺ cultures and nine times higher than in the CD34⁺38⁺33^dim cells (data not shown). The CD34⁺38⁺33^dim subpopulation displayed the highest B cell and NK progenitor content, whereas the CD34⁺38⁺33⁺ fraction differentiated in CD14/15⁺ myeloid cells with very few NK cells. These in vitro data suggest that the CD34⁺38⁺33⁺ fraction contains few if any multipotent precursors and mainly contains myeloid progenitors, whereas the 34⁺38⁺33^dim fraction contains mainly lymphoid progenitor cells. The CD34⁺38^–33⁺ fraction contains lymphoid as well as myeloid precursors and sustained CD34⁺ cell generation (Fig. 3A). Next, we attempted to localize the population with rSRA within one or more of these three subpopulations by injecting these cell populations into NOD/SCID mice. Mice were sacrificed 2 weeks after injection, and BM was analyzed (Fig. 3B). Only CD34⁺38^–33⁺ cells were able to reconstitute mice and to maintain CD34⁺ cells in the BM, demonstrating that rSRA was confined to the CD34⁺38^–33⁺ cell population. No activity was found in the CD34⁺38⁺ cell populations. The difference in chimerism between CD34⁺38⁺33^dim and CD34⁺38^–33⁺ and between CD34⁺38⁺33⁺ and CD34⁺38^–33⁺ was significant (p < .001). To exclude the possibility that the absence of engraftment was due to the incapacity to home to the BM, the three cell populations were injected immediately in the BM cavity by IF injection [4]. Using this technique, similar results were obtained as by IV injection: only the CD38^– fraction resulted in chimerism above 0.1% in the injected femur (data not shown). After IF injection of both CD34⁺38⁺ subfractions, however, enough human cells could be detected in the injected femur for phenotypic analysis (Fig. 3C). CD34⁺38⁺33^dim cells generated relatively more CD10⁺ B cells and fewer myeloid cells than CD34⁺38^–33⁺ and CD34⁺38⁺33⁺ fractions, whereas the CD34⁺38⁺33⁺ fraction was biased to the myeloid lineage. In conclusion, the majority of the CD34⁺ cells generated after in vitro expansion were CD38⁺ myeloid and lymphoid precursor cells devoid of rSRA, whereas rSRA was confined to the CD34⁺CD38^–CD33⁺ subpopulation.

View larger version (25K): [in this window] [in a new window]   Figure 3. In vitro differentiation potential and repopulating activity of CD34+ subpopulations. (A): In vitro differentiation potential. The cells were stained with human CD45 and CD34 to detect progenitor cells, a mixture of CD14 and CD15 to detect myelomonocytic cells, CD19 to detect B-lineage committed cells, and CD56 and CD3 to detect natural killer cells. (B): Percentage of chimerism after intravenous injection of CD34+38+33dim (n = 6), CD34+38+33+ (n = 6), and CD34+38–33+ (n = 8). Data are pooled from three experiments. Equal numbers of cells (between 1.25 x 105 and 7.50 x 105 cells) were injected per experiment. (C): Phenotypic analysis of injected femur after intrafemoral injection of indicated cells, performed as in Figure 1C. Equal numbers of cells (between 105 and 1 x 105) were injected per experiment. Data for CD34+38+33dim (n = 6), CD34+38+33+ (n = 5), and CD34+38–33+ (n = 5) were pooled from two independent experiments.

Functional Analysis of CD34⁺38^–33⁺ Subpopulations
As observed in Figure 2, CD34⁺38^– cells are heterogeneous for CD45RA and CD90. Positive and negative fractions were sorted, and their differentiation potential was assessed in vitro. As shown in Figure 4, little expansion without differentiation of CD34⁺ cells was observed in the CD45RA⁺ and CD90^– subpopulations after 2 weeks of MS5 culture. On the other hand, CD34⁺ expanded in the minor CD45RA^– and CD90⁺ subpopulations. Both the CD45RA⁺ and CD90^– fraction contained a mixture of myeloid and lymphoid precursors and/or multipotent precursors as demonstrated the analysis of the differentiation markers. Statistical analysis demonstrated significant difference (p < .05) for percentage of CD34⁺ and percentage of CD19⁺ in both CD45RA^–/+ and CD90^–/+ fractions.

View larger version (17K): [in this window] [in a new window]   Figure 4. Subfractionation of the expanded CD34+38–33+ fraction based on CD45RA and CD90 expression. Sorted fractions were differentiated in vitro toward natural killer (NK) cells, B cells, and myeloid cells. After 2 weeks, the cells were stained with human CD45 and CD34 to detect progenitor cells, with a mixture of CD14 and CD15 to detect myelomonocytic cells, with CD19 to detect B-lineage committed cells, or with CD56 and CD3 to detect NK cells. Results are representative of two independent experiments performed in duplicate, both for MS5 and serum-free cultures.

View larger version (22K): [in this window] [in a new window]   Figure 5. Severe combined immunodeficient repopulation by CD34+38– subpopulations: CD90–/+ and CD45RA–/+. (A, B): Percentage of chimerism after intravenous injection of indicated subpopulations. Between 5 x 104 and 105 CD45RA– or CD45RA+ cells were injected and 105 CD90– or CD90+ cells. (C, D): Phenotypic analysis of bone marrow as described in Figure 1C. Shown are mean values of the three experiments.

View larger version (23K): [in this window] [in a new window]   Figure 6. Intrafemoral injections of CD90–/+ and CD45RA–/+ subpopulations. (A, B): Percentage of chimerism in mice injected with indicated CD34+CD38– subpopulations. Between 8 x 104 and 1.5 x 105 cells were injected. Equal cell numbers were injected per experiment for all four fractions. LF was not injected. (C, D): Phenotypic analysis of the injected femurs as described in Figure 1C. (A, B): n = 3. (C, D): Mean values of the three experiments. Abbreviations: LF, left femur; RF, right femur.

Conclusion
Our results lead to the conclusion that rSRA after culture is confined to the CD34⁺38^–33⁺ fraction, independent of the cultured condition used (MS5 or SF cultured). The bulk of the rSRA is restricted to a minor CD34⁺CD38^–CD90⁺CD45RA^– cell fraction. rSRA of the CD34⁺CD38⁺CD90^–/CD45RA⁺ cells is limited and consists to a large degree of B cell precursor activity. Moreover, we show that lack of rSRA is not due to homing difficulties, as IV and IF injections gave similar results.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
In this study, we have demonstrated that rSRA is confined to a small subset of the expanded CD34⁺ cells. These cells are CD34⁺38^–90⁺45RA^–. rSRA of the remaining CD34⁺ cells is very limited and consists to a large degree of B cell precursor activity.

We chose two very different culture conditions for the characterization of the cells responsible for rSRA after culture. MS5 stromal cell culture was chosen because this condition has been shown to impair differentiation in cytokine expansion cultures, especially of cells that have divided more than twice during culture [26, 31]. In addition, the MS5 cells support both lymphoid and myeloid differentiation of stem and precursor cells [32]. No reports were found, however, of the effect of MS5 culture on SRA. In our hands injection of MS5 mixed stem cells resulted in the immediate death of the animal, probably due to obstruction of the pulmonary vascular bed by these large cells. We therefore removed these murine cells by flow cytometric cell sorting. The depleted fraction could then be analyzed for rSRA without loss of experimental animals. Despite the substantial increase in CD34⁺ cell numbers and the reported increases in LTC-IC, MS5 culture resulted in lower percentages of chimerism than when fresh cells were injected.

The other culture condition consisted of serum-free medium and high concentrations of cytokines. SF medium has been developed for stem cell expansion, as serum may result in loss of SRCs [33]. Numerous reports can be found in literature stating a minor increase after culture in SRA as determined by percentage chimerism and SRC as determined by limiting dilution analysis [12, 15, 18, 34, 35]. However, very few studies report rSRA after culture in a suitable NOD/SCID model with blockade of NK activity. We found no increased chimerism early after transplantation. We did not perform limiting dilution analysis or clonal analysis and therefore cannot conclude that the number of stem cells responsible for early repopulation have decreased. In agreement with our results, Mazurier et al. [10] reported a severe decrease in rSRA at 3 weeks by UCB CD34⁺ cells cultured for 4 days under conditions very similar to our SF cultures. They showed that the decreased activity was due to decreased stem cell number, as they observed a reduction in clones participating in reconstitution.

We have chosen two very different culture conditions for the characterization of the cells responsible for rSRA after culture. In this way, we were able to show that independent of the culture conditions only a minor fraction of the CD34⁺ cells had rSRA and that the phenotype of these cells was similar. Nevertheless, we focused most of the experiments on cells cultured on MS5 because the phenotypic diversity was much larger. The major difference between SF and MS5 cultured CD34⁺ cells was the expression pattern of CD38. Since we could generate CD34⁺38⁺ cells from fresh CD34⁺38^– cells and not vice versa, we concluded that in MS5 cultures, CD38 expression may still correlate inversely with stem cell activity. Dorrell et al. [11] cultured CD34⁺38⁺ cells in low serum with cytokines and obtained predominantly CD34⁺38^– cells. We observed, as they did, an almost complete loss of CD38 expression in SF cultures, whereas in MS5 and serum-containing cultures, CD38 expression is constant. In both culture conditions, virtually all CD34⁺ cells were CD33⁺, as described [11]. In MS5 culture, however, a small but distinct CD33^dim population could be distinguished. This fraction was CD38⁺ and partially CD7⁺. Consistent with this phenotype, in vitro testing showed that this population was enriched for B cell precursor activity but devoid of rSRA. All cells with both lymphoid and myeloid repopulating activity were CD33 brightly positive. Although CD33 is considered an early myeloid marker, CD33 is also expressed in the thymus on early lymphoid precursors [36, 37], and we show in this paper that after culture, CD34⁺38^–90⁺45RA^– cells are homogeneously CD33⁺ and contain both myeloid and lymphoid rSRA.

In vitro differentiation and in vivo repopulation assays demonstrated important differences between CD90^–/+ and between CD45RA^–/+ fractions. Both CD90⁺ and CD45RA^– fractions showed CD34⁺ expansion in vitro and were responsible for the bulk of rSRA. The CD90^– and CD45RA⁺ fractions, on the other hand, resulted in vitro in very low expansion of CD34⁺ cells but a relative high production of B cells, which has been reported of CD45RA⁺ cells [38]. In vivo, these latter fractions gave minor repopulation with a higher percentage of CD10⁺ pre-B cells than their counterparts.

The major finding reported here is that only CD34⁺CD38^–CD90⁺CD45RA^– cells were able to reconstitute NOD/SCID mice rapidly. Using fresh cells, we and others reported that NOD/SCID repopulating activity at 6–8 weeks and even more pronounced at 12 weeks is initiated mainly by CD38^– cells (3% of the CD34 cells). In contrast, the majority of the human cells detectable shortly after transplantation of CD34⁺ cells are derived from CD38 positive cells [4, 8, 16]. These data were refined by Mazurier et al. [4], who showed that the CD38^– cells repopulated at 6–12 weeks but not at 2 weeks, whereas the CD38^low cells had both activities. CD38^high cells were devoid of both repopulating activities. It is therefore difficult to conclude that the data on fresh cells are different from our data on cultured cells, since we set the cutoff for CD38 to include CD38^– and CD38^low cells (33% of CD34⁺ cells are negative). In addition, we could show in preliminary experiments that also fresh sorted CD34⁺CD38^–/low CD90⁺(CD45RA^–) cells display rSRA upon injection in NOD/SCID mice (data not shown). These data are in agreement with those of Mazurier et al. [4] and show that fresh as well as cultured CD34⁺CD38^–CD90⁺CD45RA^– cells bear rSRA.

It was reported by Danet et al. [22] that LT-SRC in cultured cells are confined to the same phenotypic population that we describe here: CD34⁺CD90⁺ fraction. CD90 is the most stable expressed surface marker during expansion, as both the amount of CD90⁺ cells and the total number of LT-SRC after culture are comparable with those of fresh cells. It is therefore possible that after in vitro expansion the majority of the stem cells have rSRA as well as LT-SRA. In addition, in murine stem cells, there is no clear distinction between early and late repopulating stem cells, as almost all LT-SRC also have ST-SRA [39]. Clonal diversity studies (human and murine) have confirmed that this classification in early and late repopulating fractions may be arbitrary, as some clones were present both early and late after transplantation [10, 40, 41].

It is hard to say whether repopulation in our animal model has any relevance to stem cell transplantation in humans. SCID and NOD/SCID are known to be receptive exclusively for LT-SRC, and as such, these models are very different from patients. Because of these discrepancies, we were the first to modify this model by injecting these mice with CD122 mAb before introduction of the human cells [5, 42]. CD122-injected mice allow engraftment of fresh CD34⁺CD38⁺ cells, which gave rise to a short wave of human cells. Recently, McKenzie et al. have compared short-term engraftment in different models via both direct IF injection and IV injection: NOD/SCID/ß2m^–/– mice and CD122-injected mice [6]. They observed that the model we introduced, mice treated with CD122, was superior to the other models with regard to engraftment of rSRC. It is therefore likely that the cell population that in our hands did not give rise to or only marginally gave rise to rSRA may behave similarly in humans. Two other arguments favor this conclusion. First, these cell populations did not engraft well after IF injection, which excludes the possibility that the capacity to home in a murine environment may be causing the defect. Second, the cells were unable to sustain generation of CD34⁺ cells in vitro on MS5 cells with early cytokines for 2 weeks.

During the course of these experiments, we focused on human CD45⁺ repopulation at 2 weeks. Patients transplanted with UCB typically show neutrophil engraftment after 3–4 weeks only. The challenge, therefore, is to obtain precursors that generate myeloid cells before that time. For this reason, some groups have tried in vitro expansion cultures. Quality control performed on these expanded cells before injection in patients usually include total cell number, CD34⁺ cell numbers, CFU-GM and BFU-E measurements and LTC-IC enumerations. As reported here, CD34⁺CD38^–CD90⁺CD45RA^– cell enumeration may be more predictive of this early activity.

	DISCLOSURES

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
We thank Caroline Collier for animal care; Dienst voor het Bloed, Rode Kruis-Vlaanderen, for the supply of UCB; and Dr. Tom Boterberg from the Department of Radiotherapy of the Ghent University Hospital for NOD/SCID irradiation. K.V. is a research assistant of the Fund for Scientific Research-Flanders. This work was supported by Fund for Scientific Research-Flanders Grant 9.0096.05 and the Research Fund of Ghent University.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 

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