The purified CD34+ cell fraction has been used for hematopoietic stem cell transplantation since they were demonstrated to have long-term reconstituting ability. Therefore, the potential effects of CD34− stem cells on the clinical course have been a major concern in recipients of CD34+-selected transplantation. To address this concern, we used an in vitro assay to determine whether transplant recipients have CD34−precursor population. Lin−CD34− cells were isolated from bone marrow cells in 11 transplant recipients including four CD34-selected transplantations, six standard bone marrow transplantations, and one T cell-depleted marrow transplantation. The frequency of the Lin−CD34− population in four CD34-enriched transplantation recipients was not different from those of normal donors or recipients of other modes of transplantation: 0.96 ± 1.01% (mean ± s.d., n = 4), 0.45 ± 0.16% (n = 6), and 0.66 ± 0.59% (n = 7), respectively. However, the Lin−CD34−population obtained from the recipients of CD34-enriched transplantation acquired neither CD34 expression nor colony-forming activity after 7 days of culture, whereas the cells from all the normal individuals and standard BMT recipients were able to differentiate into CD34+ cells accompanied by the emergence of colony-forming activity. We conclude that recipients of CD34-enriched transplantation appear to have defects in their CD34− precursor population. The clinical significance of these defects will be determined in a life-long follow-up of these patients. Bone Marrow Transplantation (2001) 28, 587–595.
Hematopoietic stem cell transplantation (HSCT) is a therapeutic procedure in which all the host components of the hematolymphoid system are reconstituted by transferred stem cells. Experimental transplantation studies in mammals, including mice, baboons and rhesus monkeys, show that a CD34+ population, corresponding to 1 to 4% of total bone marrow (BM) or mobilized peripheral blood (mPB) cells, can provide durable donor-derived long-term host hematopoietic reconstitution.1,2,3 On the basis of these findings, CD34 has been widely used as a stem and progenitor cell marker, and hence clinical autologous CD34+ stem cell transplantation (CD34+-SCT) has been performed for tumor purging.4 To reduce the incidence and severity of acute graft-versus-host disease (GVHD), several groups, including ourselves, started allogeneic transplantation using selected CD34+ BM or mPB cells to eliminate the T lymphocytes in histoincompatible cases.5,6,7,8,9 The definitive proof that the CD34+ population retains the hematopoietic stem cell (HSC) activity, however, awaits the life-long outcomes of clinical HSCT trials using CD34+ cell allografts.
Recently class of HSCs other than CD34+ cells has been demonstrated. In adult mouse bone marrow cells, a CD34−/low fraction contained long-term reconstituting activity, rather than a CD34high fraction.10,11,12,13 These cells were referred to as CD34− HSCs. The presence of human CD34− HSCs was also demonstrated by using xenogeneic transplantation systems such as a human/sheep model or NOD/SCID mice. Zanjani et al14 and Bhatia et al15 showed that the transplantation of human CD34− populations resulted in long-term, multi-lineage human cell engraftment in a human/sheep model and NOD/SCID mice, respectively. Therefore, there is heterogeneity of HSC with respect to CD34 expression in humans as well as in mice.
Since the first demonstration of CD34− stem cells, one of the biggest concerns in the field of clinical hematology has been the presence or the absence of CD34− stem cells in the recipients of CD34+-SCT and its consequence on the clinical course after transplantation. If indeed human HSCs are present in the CD34− marrow population, then enriching the CD34+ cells will result in the loss of at least a portion of HSCs.16
So far, only functional assays such as xenogeneic transplantation systems are available to assess CD34− HSCs since no specific markers for CD34− stem cells have been discovered. We have recently established an in vitro system in which CD34+ HSCs can be induced from a CD34− cell population. This system is useful for detecting the CD34− precursor population of CD34+ cells.17,18 Here we evaluated the CD34− cell population from four recipients of the CD34+-SCT along with their donors and those of other modes of HSCT using the in vitro system.
Materials and methods
Patients characteristics and clinical outcomes
Eleven patients who were given allogeneic stem cell transplantation between October 1992 and May 1999 were enrolled in this study. Four patients received CD34+-selected BM or PB grafts from their two or three HLA-antigen mismatched parents. One patient received T cell-depleted (TCD) marrow from her two HLA antigen mismatched brother. Six patients received unmanipulated BM grafts from their HLA-matched siblings. Age, sex, body weight and disease of the patients as well as age, relation, body weight and HLA compatibility of the donors, pre-conditioning regimen and GVHD prophylaxis are shown in Tables 1 and 2. Informed consent was obtained from all of the patients and donors or their guardians.
Grafts and cell processing
Transplanted cells and transplantation outcomes are summarized in Table 3. CD34+ cell selection was done using the Isolex-50 system (Baxter Healthcare, Irvine, CA, USA). CD34+ purity in the four grafts was 93.7%, 96.3%, 97.3% and 81.3%. Transplanted CD34+ cells ranged from 2.43 to 26.6 × 106/kg, and CD3+ cells from 0.77 to 6.29 × 104/kg. TCD was done using an immunoadsorption flask coated with CD5 and CD8 antibodies. CD34+ purity was 7.26%, and 1.42 × 106/kg of CD34+ cells were transplanted. The number of mononuclear cells in the unmanipulated BM grafts ranged from 2.56 to 3.73 × 108/kg. The frequency of CD34+ and CD3+ cells in the grafts was not measured in these unmanipulated grafts.
Engraftment was achieved in all patients. The days of hematological recovery ranged from 10 to 25 for neutrophils (500/μl) and from 23 to 129 for platelets (50 000/μl). There was no difference in the days of engraftment between the CD34+ SCT recipients and the recipients of other forms of graft. The post-transplant courses of these patients were generally uneventful. Acute GVHD was absent or mild, and limited chronic GVHD was present in one of four CD34+ SCT recipients and two of seven unmanipulated BMT recipients. There were no major infectious complications or disease recurrence in those patients between transplant and the time of bone marrow sample collection for this study. Immune recovery was more delayed in CD34+ SCT recipients, but immune function returned to the normal range in most of the patients at the time of sample collection. Thus, both groups were comparable post transplant. All patients are alive and well at the time of the BM aspiration for this study. The interval between transplantation and BM aspiration ranged from 5 months to 7 years. Bone marrow and peripheral blood were all normal at the time of this study.
Bone marrow cells were obtained from patients and healthy donors according to the guidelines approved by the Tokai University Committee on Clinical Investigation. Mononuclear cells (MNCs) were stained with fluorescein isocyanate (FITC)-conjugated antihuman CD45 (T-200), phycoerythrin(PE)-conjugated antihuman CD2 (39C1.5), CD3 (UCHT1), CD7 (M-T701), CD14 (LeuM3), CD16 (3G8), CD19 (4G7), CD20 (2H7), CD33 (WM53), CD41 (P2), CD56 (N901), and glycophorin A (KC16). Stained cells were sorted on a FACSVantage (Becton Dickinson, San Jose, CA, USA) equipped with an argon laser tuned to 488 nm. Sorted lineage marker-negative CD45-positive (Lin−CD45+) cells were restained with PE-conjugated antihuman CD34 (581), and a Lin−CD34− fraction was obtained by second sorting. The viability of sorted cells was more than 95% as determined by trypan-blue dye exclusion.
In vitro culture system
The hematopoietic supportive stromal cell line HESS-5 and stroma-dependent culture conditions were reported previously.19,20 Briefly, Lin−CD34− cells were plated on to pre-established irradiated HESS-5 monolayers in StemProTM-34SFM (GibcoBRL, Grand Island, NY, USA) supplemented with StemProTM-34 nutrient supplement, 5% FCS and cytokines. The final concentrations of cytokines were as follows: TPO, 300 ng/ml; FL, 300 ng/ml; SCF, 300 ng/ml; G-CSF, 10 ng/ml; IL-3, 10 ng/ml; IL-6, 10 ng/ml. Human IL-3, G-CSF, SCF, and TPO were a generous gift from the Kirin Brewery Co Ltd (Tokyo, Japan).
Flow cytometric immunophenotyping
Aliquots of sorted cells or cultured cells were suspended in EDTA-BSA-PBS and incubated with mouse IgG (Inter-Cell Technologies, Hopewell, NJ, USA) to block any nonspecific binding. The cells were then reacted for 15 min with anti-human CD34-FITC (QBEND10) and CD45-PE. Unbound antibodies were removed by two washes, and then the cells were resuspended in EDTA-BSA-PBS. Stained cells were then passed through a nylon mesh filter and subjected to two-color flow cytometric analysis. Gating on the lymphoid region was used to exclude stromal cells by size and granularity. The cells labeled with FITC- and PE-conjugated mouse isotype-matched antibodies were used as a control. The surface markers of the cells were analyzed by FACSCalibur using Cell Quest software (Becton Dickinson).
RT-PCR and PCR
To detect contaminating CD34+ cells in the sorted samples, total RNA was prepared from 1 × 103 freshly sorted cells using ISOGEN (Nippon Gene, Toyama, Japan) and reverse-transcribed using oligo dT primer and RAV-2 reverse transcriptase (Takara, Otsu, Japan) as described previously.17 The PCR conditions were optimized for each primer set to maintain amplification in the linear range. The products were separated on a 1.0% agarose gel, transferred to membranes and then hybridized with probes labeled by using ECL labeling and a Detection System (Amersham, Buckinghamshire, UK). The sensitivities of RT-PCR and PCR were 10−5 and 10−4, respectively. To determine the origin of the CD34−Lin− fraction, a variable number of tandem repeats (VNTR) locus (D1S80) and short tandem repeat (SRT) locus (ACTBP2) was amplified by PCR with specific primers already reported.21
The CFU-C assay was performed as described previously.20 All cultures were done in triplicate and the number of CFU-C was scored on day 14 of culture. The colony types were determined by in situ observations using an inverted microscope.
Lin−CD34− cells in patients after HSCT and normal donors
The frequency of Lin−CD34− cells among all nucleated BM cells was 0.96 ± 1.01% (mean ± s.d., n = 4) in the CD34+ SCT recipients, whereas the frequency was 0.66 ± 0.59% (n = 7) in other recipients and 0.45 ± 0.16% (n = 6) in normal donors. There was no significant difference in the frequencies of Lin−CD34− fractions among these three groups. However, there was a striking difference in CD45 expression in the Lin− fraction between the CD34+ SCT recipients and other groups (Figure 1).
Representative data are shown in Figure 1a and c. The recipient depicted in Figure 1a (patient 139) received a graft from the donor in Figure 1c (patient's mother). More than 90% of the Lin− cells belonged to the CD45high fraction in patients 139 (Figure 1a), 220, 232 and 325 (not shown), whereas 90% of Lin− cells belonged to the CD45mid fraction in BM cells from the other recipients (Figure 1b) and normal donors (Figure 1c). Furthermore, when the sorted Lin− cells were restained with CD34, most of the CD34+ cells were CD45mid.
A functional assay is required to detect CD34− stem cells within this fraction, due to the lack of a positive marker for CD34− stem cells. For that purpose, as shown in Figure 1, more than 96% pure fraction of Lin−CD34− cells was sorted and used for subsequent experiments.
Sorted cells in all cases were analyzed to determine CD34 mRNA expression by using a very sensitive semi-quantitative RT-PCR, and the absence of CD34 mRNA was confirmed (Figure 2). The results indicate that the sorted fractions are not only negative for the surface expression of CD34 antigen, but also negative for CD34 gene expression, which makes the possibility of CD34+ cell contamination extremely unlikely. The origin of the cells was also determined by PCR analysis of the genetic polymorphism at a VNTR locus (D1S80) and SRT locus (ACTBP2) (Figure 3). The disappearance of a 256-bp band in No. 139, 177 bp in No. 220, and a 264-bp band in No. 232 demonstrated that they were derived from the donor. The disappearance of a PCR band derived from HLA DR8 in No. 325 confirmed that the cells were derived from the donor (data not shown).
Cell growth and CD34 induction in culture
The viability and proliferation of the Lin− CD34− cells were measured in the stroma-dependent culture system which contained a combination of cytokines: human TPO, FL, SCF, G-CSF, IL-3 and IL-6.17 The viability of cells was maintained throughout the culture period. The number of cells from healthy donors and recipients of transplants other than CD34+ SCT was increased by 4.2 ± 2.0-fold (n = 13) and 16.4 ± 7.8-fold (n = 13) after 7 and 14 days, respectively, in culture. On the other hand, no proliferation was observed in Lin− CD34− cells from the CD34+ SCT recipients, and they gradually lost viability in this culture system: 0.45 ± 0.15-fold (n = 4) and 0.25 ± 0.18-fold (n = 4) after 7 and 14 days, respectively.
We then analyzed the surface expression of CD34 in cultured cells. Representative data were shown in Figure 4. Definite expression of CD34 was detected in both healthy donors and recipients other than CD34+ SCT (patient Nos 120, 168, and 231). The mean frequency of CD34+ cells after 7 days culture was 21.8 ± 13.8% (n = 7) in recipients not given CD34+ SCT and 39.0 ± 19.9% (n = 6) in normal donors. The cells expressed CD45, confirming that they were of human hematopoietic and not of stromal origin. As we have demonstrated previously using cord blood, CD34+ cells were derived from the CD45mid population.17 On the other hand, cells from the CD34+ SCT recipients (patient Nos 139, 220, 232 and 325) did not show any CD34 expression. These results suggest that the Lin−CD34− cells from the CD34+ SCT recipients either did not contain the precursor population of CD34+ cells or that the cultured cells had impaired CD34 expression. Therefore, to test these two possibilities, the functional capacity of cultured cells was examined by clonogenic assay.
Clonogenic ability of Lin−CD34− cells before and after culture
To evaluate the relationship of CD34 induction on the cell surface and clonogenic ability of Lin−CD34− cells, CFU-C output was determined before and after culture. Freshly sorted Lin−CD34− cells did not possess any hematopoietic colony-forming capacity. As shown in Figure 5, the number of CFU-C per 1000 cells was 13.2 ± 3.2 (n = 7) and 24.8 ± 12.9 (n = 6) after 7 days of culture in recipients not given CD34+ SCT and normal donors. However, in the CD34+ SCT recipients, no CFUs were induced after culture (n = 4), whereas sorted Lin−CD34+ cells had CFU activity as expected. Thus, the colony-forming activity in the cultured cells was positively correlated with the extent of CD34 expression induced from Lin−CD34− cells (r = 0.93). These data suggest that there is a strong relationship between the expression of CD34 and the in vitro response to cytokines that drive proliferation and myeloid and erythroid differentiation. The inability of CD34+ cell induction from Lin−CD34− cells in the three recipients of CD34+ SCT suggest the absence of CD34− stem cells in their hematopoietic system.
In this study we present data suggesting the absence of CD34− stem cells in the recipients of CD34+ SCT by using an in vitro assay. Accumulated data indicate that CD34− stem cells are more primitive than CD34+ stem cells both in human and rodents.10 In adult mice, single murine Lin−c-kit+Sca-1+CD34− cells, not CD34+ cells, transplanted into lethally irradiated mice could sustain long-term multilineage engraftment.10 The different kinetics in the two populations was also reported in a human/sheep model, in which human cell activity in animals reconstituted by CD34+ cells was completely exhausted by treatment with four cycles of human IL-3/GM-CSF, whereas no significant effect was noted in animals reconstituted by CD34− cells.22 Thus, to clarify the clinical significance of human CD34− stem cells, the hematopoietic system in the recipients of CD34+ SCT was analyzed.
Studies on human CD34− stem cells, however, have been hindered by the lack of a positive marker, comparable to the Sca-1 and c-kit in mice and thus functional assays such as xenotransplantation have been used so far. Bhatia et al reported that the frequency of SRC is one in 125 000 Lin−CD34− cells, while other reports detected no SRC in Lin−CD34− fraction of cells.15,23,24 Compared to the frequency of one SRC in 617 CD34+CD38− cells, the sensitivity of the xenogeneic transplantation system is not high enough to assess the CD34− HSCs in clinical samples.25
We have recently established a xeno-coculture system in which human CD34+ HSCs were induced from the CD34− cell population. In this study, we used this system as a means to assess CD34− stem cells which were induced to express CD34.17 The induction of CD34 expression was recently reported to be related to the activation of HSC in mice.13 If this also applies to humans, CD34 induction is a useful functional characteristic of CD34− HSC. Based on this evidence, the absence of CD34 induction accompanying CFUs in the CD34+ SCT recipients indicates the absence of CD34− HSCs rather than the functional impairment of CD34− HSCs. Moreover, this in vitro system should be useful to assess CD34− HSC in other clinical settings, such as leukemia and other hematopoietic disorders.
On the other hand, our data also demonstrated that the transplanted CD34+ cells have long-term multilineage reconstituting ability, since the hematopoietic system reconstituted by donor CD34+ cells has persisted for as long as 7 years in patient 139 and 4 years in patient 232, without any hematological complications. Recently KDR (also known as vascular endothelial growth factor receptor 2; VEGFR2) positive cells, which comprise 0.1 to 0.5% of human CD34+ cells, were demonstrated to be pluripotent HSC.26 Therefore this fraction of CD34+ cells may be responsible for the long-term reconstituting ability in the CD34+ SCT recipients and should be further examined in clinical CD34+ SCT.
In this report we compared long-term surviving CD34+ SCT recipients with T cell-depleted or unmanipulated bone marrow recipients. The compositions of each groups are heterogenous in terms of underlying diseases of the patients, ages of the patients and the donors, conditioning regimen and GVHD prophylaxis. Furthermore, CD34+ SCT recipients are very different from unmanipulated BM recipients in HLA disparity and transplanted T cell doses, although actual T cell numbers were not counted in the unmanipulated bone marrow. Therefore, we cannot exclude the possibility that HLA disparity and/or absence of T cells in the graft might have contributed to the reduced CD34− HSC reconstitution in CD34+ SCT recipients. However, CD34− stem cell function was demonstrated in one patient who was given TCD bone marrow from her two HLA antigen mismatched brother, suggesting that such a possibility is less likely.
Recently, the reversibility of CD34 expression in stem cells was reported in mice. Sato et al13 transplanted CD34+ cells from the BM of 5-FU-treated mice into lethally irradiated hosts and found that these CD34+ cells reverted to the CD34− phenotype as the marrow attained steady state phase 3 months later. If this is also the case for human HSC, stem cells in the CD34+ selected graft may revert to CD34− phenotype, but this seems to contradict our results. This reversion was seen in the activated CD34+ stem cells induced by 5-FU-treatment, and stem cells in steady state hematopoiesis were CD34− such as in normal donors, which may explain the discrepancy.
Now that we know that the Lin−CD34− fraction contains the precursor of CD34+ cells, transfusion of this fraction to the recipient, in addition to the CD34+ fraction, is expected to improve the engraftment and clinical outcome rather than worsen it. We have recently developed a filter to enrich these fractions of cells and this system is expected to be practical enough for clinical use.27
In conclusion, we have shown that the Lin−CD34− fraction of marrow cells in the CD34+ SCT recipients was unable to acquire CD34 expression and CFU in an in vitro system. This finding suggests the absence of CD34− stem cells in the CD34+ SCT recipients. The clinical significance of this finding requires further observation of their clinical course. Cotransfusion of a Lin−CD34− fraction, in addition to CD34+ cells, can be expected to improve the clinical outcome in histoincompatible CD34+ SCT.
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We thank Hideyuki Matsuzawa for his excellent technical assistance, Shizuko Imai for secretarial work and the members of Tokai CBSC Study Group for their assistance. The authors also wish to thank KIRIN Brewery Co. Ltd. for supplying various growth factors. This study was supported by The Japan Society for the Promotion of Science (JSPS) grant no. JSPS-RFTF97 I 00201 and a research grant of the science Frontier Program from the Ministry of Education, Science, Sports and Culture of Japan.
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Kato, S., Ando, K., Nakamura, Y. et al. Absence of a CD34− hematopoietic precursor population in recipients of CD34+ stem cell transplantation. Bone Marrow Transplant 28, 587–595 (2001). https://doi.org/10.1038/sj.bmt.1703186
- CD34-enriched transplantation
- CD34− precursor population
- hematopoietic stem cells
- bone marrow stroma cells
Experimental Hematology (2007)
Leukemia Research (2005)
International Journal of Hematology (2002)
Nature Immunology (2002)