Spontaneous and induced osteoclastogenic behaviour of human peripheral blood mononuclear cells and their CD14(+) and CD14(-) cell fractions

Objectives: Osteoclasts are descended from the CD14⁺ monocyte/macrophage lineage, but influence of other haematopoietic cells on osteoclastic commitment of their precursors has remained poorly understood. In this study, osteoclastogenic behaviour of peripheral blood mononuclear cells (PBMC) and their CD14⁺ and CD14^? subpopulations has been accessed, in the absence or presence of M‐CSF and RANKL. Materials and Methods: Cell cultures were characterized for presence of actin rings and vitronectin and calcitonin receptors, TRAP activity and calcium phosphate resorbing activity, expression of osteoclast‐related genes and secretion of M‐CSF and RANKL. Results: In the absence of growth factors, PBMC and CD14⁺ cultures had some degree of cell survival, and some spontaneous osteoclastogenesis was observed, only on cultures of the former. Supplementation with M‐CSF and RANKL significantly increased osteoclastogenic behaviour of cell cultures, particularly CD14⁺ cell cultures. Nevertheless, PBMC derived a higher degree of osteoclastogenesis, either as absolute values or after normalization by protein content. It was observed that unlike CD14⁺ cells, PBMC were able to express M‐CSF and RANKL, which increased following growth factor treatment. Also, expression of TNF‐α, GM‐CSF, IL‐1β, IL‐6 and IL‐17 was higher in PBMC cultures. Finally, CD14^? cultures exhibited limited cell survival and did not reveal any osteoclast features. Conclusions: Results show that although osteoclastic precursors reside in the CD14⁺ cell subpopulation, other populations (such as CD14^? cells) derived from PBMC, have the ability to modulate osteoclastogenesis positively.     

Rejuvenation of neutrophils and their extracellular vesicles is associated with enhanced aged fracture healing

Tissue repair is negatively affected by advanced age. Recent evidence indicates that hematopoietic cell‐derived extracellular vesicles (EVs) are modulators of regenerative capacity. Here, we report that plasma EVs carrying specific surface markers indicate the degree of age‐associated immunosenescence; moreover, this immunosenescence phenotype was accentuated by fracture injury. The number of CD11b⁺Ly6C^intermediateLy6G^high neutrophils significantly decreased with age in association with defective tissue regeneration. In response to fracture injury, the frequencies of neutrophils and associated plasma EVs were significantly higher in fracture calluses than in peripheral blood. Exposure of aged mice to youthful circulation through heterochronic parabiosis increased the number of neutrophils and their correlated Ly6G⁺ plasma EVs, which were associated with improved fracture healing in aged mice of heterochronic parabiosis pairs. Our findings create a foundation for utilizing specific immune cells and EV subsets as potential biomarkers and therapeutic strategies to promote resilience to stressors during aging.     

Concomitant overexpression of triple antioxidant enzymes selectively increases circulating endothelial progenitor cells in mice with limb ischaemia

Endothelial progenitor cells (EPCs) are a group of heterogeneous cells in bone marrow (BM) and blood. Ischaemia increases reactive oxygen species (ROS) production that regulates EPC number and function. The present study was conducted to determine if ischaemia‐induced ROS differentially regulated individual EPC subpopulations using a mouse model concomitantly overexpressing superoxide dismutase (SOD)1, SOD3 and glutathione peroxidase. Limb ischaemia was induced by femoral artery ligation in male transgenic mice with their wild‐type littermate as control. BM and blood cells were collected for EPCs analysis and mononuclear cell intracellular ROS production, apoptosis and proliferation at baseline, day 3 and day 21 after ischaemia. Cells positive for c‐Kit⁺/CD31⁺ or Sca‐1⁺/Flk‐1⁺ or CD34⁺/CD133⁺ or CD34⁺/Flk‐1⁺ were identified as EPCs. ischaemia significantly increased ROS production and cell apoptosis and decreased proliferation of circulating and BM mononuclear cells and increased BM and circulating EPCs levels. Overexpression of triple antioxidant enzymes effectively prevented ischaemia‐induced ROS production with significantly decreased cell apoptosis and preserved proliferation and significantly increased circulating EPCs level without significant changes in BM EPC populations, associated with enhanced recovery of blood flow and function of the ischemic limb. These data suggested that ischaemia‐induced ROS was differentially involved in the regulation of circulating EPC population.     

CD161 Expression Defines a Th1/Th17 Polyfunctional Subset of Resident Memory T Lymphocytes in Bronchoalveolar Cells

Alveolar resident memory T cells (T_RM) comprise a currently uncharacterized mixture of cell subpopulations. The CD3⁺CD161⁺ T cell subpopulation resides in the liver, intestine and skin, but it has the capacity for tissue migration; however, the presence of resident CD3⁺CD161⁺ T cells in the bronchoalveolar space under normal conditions has not been reported. Bronchoalveolar cells (BACs) from healthy volunteers were evaluated and found that 8.6% (range 2.5%-21%) of these cells were CD3⁺ T lymphocytes. Within the CD3⁺ population, 4.6% of the cells (2.1–11.3) expressed CD161 on the cell surface, and 74.2% of the CD161⁺CD3⁺ T cells expressed CD45RO. The number of CD3⁺CD161⁺ T cells was significantly lower in the bronchoalveolar space than in the blood (4.6% of BACs vs 8.4% of peripheral blood mononuclear cells (PBMCs); P<0.05). We also found that 2.17% of CD4⁺ T lymphocytes and 1.52% of CD8⁺ T lymphocytes expressed CD161. Twenty-two percent of the alveolar CD3⁺CD161⁺ T lymphocytes produced cytokines upon stimulation by PMA plus ionomycin, and significantly more interferon gamma (IFN-γ) was produced compared with other cytokines (P = 0.05). Most alveolar CD3⁺CD161⁺ T cells produced interleukin-17 (IL-17) and IFN-γ simultaneously, and the percentage of these cells was significantly higher than the percentage of CD3⁺CD161⁻ T cells. Moreover, the percentage of alveolar CD3⁺CD161⁺ T lymphocytes that produced IFN-γ/IL-17 was significantly higher than those in the peripheral blood (p<0.05). In conclusion, Th1/Th17-CD3⁺CD161⁺ T_RM could contribute to compartment-specific immune responses in the lung.     

Shared circulation in parabiosis leads to the transfer of bone phenotype from gld to the wild-type mice

We have previously shown that mice with generalised lymphoproliferative disorder (gld) have increased bone mass in addition to autoimmune disease characterised by the accumulation of double negative (dn) T lymphocytes (CD3(+)CD4(-)CD8(-)CD45R(+)). To further explore the association of the immune disorder with the bone phenotype of gld mice, we established parabiotic circulation between gld and wild-type animals (C57BL/6, B6). One week after the surgery, the proportion of dn T lymphocytes increased in peripheral blood, bone marrow, spleen, and lymph nodes of wild-type members of the B6-gld parabiotic pair and decreased in tissues of gld pair members. The mixing of cells continued during four weeks of parabiosis. Number of osteoclast-like (OCL) cells in bone marrow cultures from a wild-type member of B6-gld parabiotic pair at the end of the first week decreased from 266+/-52 to 120+/-5OCL/cm(2), P<0.05, comparable with gld mice (99+/-21OCL/cm(2)), while the number of osteoblast colonies did not change. After four weeks, number of OCL cells formed from the bone marrow of B6 parabiotic mice was still similar to the number of OCL cells in their gld counterparts (150+/-18 and 131+/-24OCL/cm(2), respectively). In addition, the number of osteoblast colonies in B6 members of B6-gld parabiotic pairs increased (from 6+/-2 to 18+/-1colonies/cm(2), P<0.05) thus resembling the cell cultures of gld mice (18+/-1colonies/cm(2)). Taken together, these data show that the circulation of cells, including dn T lymphocytes established by parabiosis confers the osteoclast and osteoblast phenotype of gld to wild-type animals.     

Circulating myeloid-derived suppressor cells: An independent prognostic factor in patients with breast cancer

Evading immune destruction is a hallmark of cancer. Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid immune cells, are thought to foster the establishment of an immunosuppressive tumor microenvironment, but it remains unclear how. This study aims to determine the levels of circulating MDSCs and their subpopulations and test their immunosuppressive functions in patients with breast cancer (BC). We analyzed the fractions of MDSCs in freshly isolated peripheral blood mononuclear cells of patients with BC and healthy donors using flow cytometry. Circulating MDSCs were further phenotyped using fluorescently labeled antihuman monoclonal antibodies. Coculture experiments revealed the effects of MDSCs on CD3⁺ T cell response. Moreover, we correlated circulating MDSC levels with clinicopathological features of patients with BC. We show that the fraction of HLA-DR ^– CD33 ⁺ MDSCs in peripheral blood is about 10-fold higher in patients with BC than in healthy control individuals. The levels of all MDSC subpopulations, including monocytic and granulocytic MDSCs, are significantly elevated. Coculture experiments of purified HLA-DR ^– CD33 ⁺ MDSCs and CD3 ⁺ T cells demonstrate that T cell proliferation is more effectively inhibited by BC patient-derived MDSCs than by healthy control MDSCs. Moreover, increased circulating MDSC levels robustly associate with advanced BC stage and positive lymph node status. By being more abundant and more effective T cell suppressors, BC patient-derived circulating MDSCs exert a dual immunosuppressive effect. Our findings pave the way to develop novel diagnostic and immunotherapeutic strategies, aimed at detecting and inhibiting MDSCs in patients with BC.     

Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast‐like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF‐β, fibronectin (FN), α‐SMA, and NG2. LPS also increased protein and gene expression levels of anti‐inflammatory COX‐2 and pro‐inflammatory IL‐6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS‐treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen‐stimulated proliferation of CD4⁺ and the ratio of CD4⁺CD25^high/CD4⁺CD25^low lymphocytes. LPS‐treated PDLSCs did not change the frequency of CD34⁺ and CD45⁺ cells, but decreased the frequency of CD33⁺ and CD14⁺ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU‐GM number. The results indicated that LPS‐activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features.     

Differences amid bone marrow and cord blood hematopoietic stem/progenitor cell division kinetics

Human hematopoietic stem/progenitor cells (HSC) isolated based upon specific patterns of CD34 and CD38 expression, despite phenotypically identical, were found to be functionally heterogeneous, raising the possibility that reversible expression of these antigens may occur during cellular activation and/or proliferation. In these studies, we combined PKH67 tracking with CD34/CD38 immunostaining to compare cell division kinetics between human bone marrow (BM) and cord blood (CB)‐derived HSC expanded in a serum‐free/stromal‐based system for 14 days (d), and correlated CD34 and CD38 expression with the cell divisional history. CB cells began dividing 24 h earlier than BM cells, and significantly higher numbers underwent mitosis during the time in culture. By d10, over 55% of the CB‐cells reached the ninth generation, whereas BM‐cells were mostly distributed between the fifth and seventh generation. By d14, all CB cells had undergone multiple cell divisions, while 0.7–3.8% of BM CD34⁺ cells remained quiescent. Furthermore, the percentage of BM cells expressing CD34 decreased from 60.8 ± 6.3% to 30.6 ± 6.7% prior to initiating division, suggesting that downmodulation of this antigen occurred before commencement of proliferation. Moreover, with BM, all primitive CD34⁺CD38^? cells present at the end of culture arose from proliferating CD34⁺CD38⁺ cells that downregulated CD38 expression, while in CB, a CD34⁺CD38^? population was maintained throughout culture. These studies show that BM and CB cells differ significantly in cell division kinetics and expression of CD34 and CD38, and that the inherent modulation of these antigens during ex vivo expansion may lead to erroneous quantification of the stem cell content of the expanded graft. J. Cell. Physiol. 220: 102–111, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization and comparison of adipose tissue‐derived cells from human subcutaneous and omental adipose tissues

Different fat depots contribute differently to disease and function. These differences may be due to the regional variation in cell types and inherent properties of fat cell progenitors. To address the differences of cell types in the adipose tissue from different depots, the phenotypes of freshly isolated adipose tissue‐derived cells (ATDCs) from subcutaneous (SC) and omental (OM) adipose tissues were compared using flow cytometry. Our results showed that CD31^?CD34⁺CD45^?CD90^‐CD105^?CD146⁺ population, containing vascular smooth muscle cells and pericytes, was specifically defined in the SC adipose tissue while no such population was observed in OM adipose tissue. On the other hand, CD31^?CD34⁺CD45^?CD90^?CD105^?CD146^? population, which is an undefined cell population, were found solely in OM adipose tissue. Overall, the SC adipose tissue contained more ATDCs than OM adipose tissue, while OM adipose tissue contained more blood‐derived cells. Regarding to the inherent properties of fat cell progenitors from the two depots, adipose‐derived stem cells (ADSCs) from SC had higher capacity to differentiate into both adipogenic and osteogenic lineages than those from OM, regardless of that the proliferation rates of ADSCs from both depots were similar. The higher differentiation capacity of ADSCs from SC adipose tissue suggests that SC tissue is more suitable cell source for regenerative medicine than OM adipose tissue. Copyright © 2009 John Wiley & Sons, Ltd.     

Influence of perioperative whole blood transfusions on lymphocyte subpopulations in patients with stage II breast cancer

Preliminary reports have suggested an adverse relationship between blood transfusion and survival after surgery in patients with solid tumour. One might postulate that from these studies, perioperative blood transfusions alter host immune defences. We therefore examined the influence of homologous whole blood transfusion on circulating lymphocyte subpopulations in transfused patients compared with non-transfused patients. Fifty-one women with Stage II breast cancer who underwent surgical procedures were studied. Patients were classified into two groups on the basis of whether or not they had received blood transfusion. The lymphocyte subpopulations were analyzed by flow cytometry before cancer surgery and three weeks after the operation. CD3⁺, CD4⁺, CD8⁺, and CD20⁺ cells as the lymphocyte subsets were quantitated using appropriate monoclonal antibodies. No significant differences between pre- and postoperative lymphocyte subset levels were seen in non-transfused patients. However, there was a statistically significant increase in the CD8⁺ cell count; decreasing CD4⁺ cell count and decreased CD3⁺ cells levels were observed in the transfused group (P<0.05). Although these early results of the study suggest that the blood transfusions could be associated with alterations in lymphocyte populations, additional studies are needed to elucidate the possible mechanism of the transfusion-induced immunological modulations.     

Autocrine VEGF signalling on M2 macrophages regulates PD‐L1 expression for immunomodulation of T cells

M2‐polarized macrophages, on one hand, can promote tumour vascularization by producing proangiogenic factors, such as vascular endothelial growth factor (VEGF). On the other hand, the expression of VEGF receptors (VEGFR) in this cell lineage was also reported. Although the function of VEGF/VEGFR axis plays a pivotal role in macrophages infiltration and angiogenesis, however, there is still lack of the direct evidence to show the role of VEGF as an autocrine operating in M2 macrophages, particularly for immunomodulation. In our study, we surprisingly discovered that M2 macrophages polarized by baicalin can simultaneously express VEGF and its receptors. Taking advantage of this unique culture system, we were able to investigate the biological activity of M2 macrophages in response to the autocrine VEGF milieu. Our results showed that the expression of programmed death‐ligand 1 (PD‐L1) on M2 macrophages was significantly up‐regulated in autocrine VEGF milieu. Through the blockade of autocrine VEGF signalling, PD‐L1 expression on M2 macrophages was dramatically down‐regulated. Furthermore, transplantation of PD‐L1⁺ M2 macrophage stimulated by autocrine VEGF into allogeneic mice significantly suppressed host CD4⁺/CD8⁺ T cells in the peripheral blood and increased CD4⁺CD25⁺ regulatory T cells in the bone marrow. In conclusion, our findings provide a novel biological basis to support the current successful strategy using combined VEGF/PD‐1 signalling blockade in cancer therapy.     

Innate-Like and Conventional T Cell Populations from Hemodialyzed and Kidney Transplanted Patients Are Equally Compromised

Clinicians are well aware of existing pharmacologically-induced immune deficient status in kidney-transplanted patients that will favor their susceptibility to bacterial or viral infections. Previous studies indicated that advanced Stage 4–5 Chronic Kidney Disease might also be regarded as an immune deficiency-like status as well, even though the mechanisms are not fully understood. Here, we analyzed the ex vivo frequency and the functional properties of both conventional and innate-like T (ILT) lymphocyte subsets in the peripheral blood of 35 patients on hemodialysis, 29 kidney transplanted patients and 38 healthy donors. We found that peripheral blood cell count of ILT cells, as iNKT (invariant Natural Killer T) and MAIT (mucosal-associated invariant T), were significantly decreased in hemodialyzed patients compared to healthy controls. This deficiency was also observed regarding conventional T cells, including the IL-17-producing CD4⁺ Th17 cells. Pertaining to regulatory T cells, we also noticed major modifications in the global frequency of CD4⁺CD25⁺Foxp3⁺ T lymphocytes, including the resting suppressive CD45RA⁺Foxp3^lo and activated suppressive CD45RA⁻Foxp3^hi T cell subpopulations. We found no significant differences between the immune status of hemodialyzed and kidney-transplanted subjects. In conclusion, we demonstrated that both ILT and conventional T cell numbers are equally impaired in hemodialyzed and kidney-transplanted patients.     

LPS administration increases CD11b<Superscript>+</Superscript> c-Fms<Superscript>+</Superscript> CD14<Superscript>+</Superscript> cell population that possesses osteoclast differentiation potential in mice

Osteoclasts are multinucleated giant cells that originate from a monocyte/macrophage lineage, and are involved in the inflammatory bone destruction accompanied by periodontitis. Recent studies have shown that osteoclast precursors reside not only in the bone marrow, but also in the peripheral blood and spleen, though the precise characteristics of each precursor have not been analyzed. We hypothesized that the number of osteoclast precursors in those tissues may increase under pathological conditions and contribute to osteoclast formation in vivo in a mouse model. To test this hypothesis, we attempted to identify cell populations that possess osteoclast differentiation potential in the bone marrow, spleen, and blood by analyzing macrophage/monocyte-related cell surface markers such as CD11b, CD14, and colony-stimulating factor-1 receptor (c-Fms). In the bone marrow, the CD11b^? cell population, but not the CD11b⁺ cell population, differentiated into osteoclasts in the presence of receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. On the other hand, in the spleen and blood, CD11b⁺ cells differentiated into osteoclasts. Interestingly, lipopolysaccharide (LPS) administration to the mice dramatically increased the proportion of CD11b⁺ c-Fms⁺ CD14⁺ cells, which differentiated into osteoclasts, in the bone marrow and spleen. These results suggest that LPS administration increases the proportion of a distinct cell population expressing CD11b⁺, c-Fms⁺, and CD14⁺ in the bone marrow and spleen. Thus, these cell populations are considered to contribute to the increase in osteoclast number during inflammatory bone destruction such as periodontitis.     

Retrovirus-mediated gene transfer into CD4+ and CD8+ human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells

Summary Studies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterialneo ^R gene. The presence of theneo ^R gene in the TIL population was demonstrated by Southern blot analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of theneo ^R gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4⁺ and CD8⁺ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4⁺ and CD8⁺ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4⁺ or CD8⁺ cells. In other experiments highly purified CD4⁺ and CD8⁺ T cell subpopulations from either TIL or PBMC could be successfully transduced with theneo ^R gene as demonstrated by Southern blot analysis and detection of the gene product neophosphotransferase activity. No such activity or vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4⁺ and CD8⁺ gene-modified TIL. Current address: Bone Marrow Transplantation, Hadassah University Hospital, 91120 Jerusalem, Israel     

Reduced naïve CD8+ T‐cell priming efficacy in elderly adults

Aging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8⁺ T‐cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8⁺ T‐cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8⁺ T‐cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8⁺ T‐cell responses specific for a model antigen. Reduced CD8⁺ T‐cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8⁺ T‐cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.     

Prediction of CD34(+) cell yield in hematopoietic cell products from children by peripheral blood CD34(+) cell counts

Background aimsPeripheral blood stem cells (PBSC) are increasingly used as an alternative to bone marrow in autologous transplantations. In adult patients, the peripheral blood CD34 ⁺ cell count is a good predictor of CD34 ⁺ cell yield in apheresis. However, the determinants of stem cell yield in the pediatric population have not been well established.MethodsWe retrospectively studied 396 apheresis procedures in 301 pediatric patients. Receiver operating characteristic (ROC) curves based on pre-apheresis peripheral blood CD34 ⁺ cell counts were generated to facilitate prediction of the optimal timing of PBSC collection. The associations between CD34 ⁺ cell yield and age and mobilization regimen were analyzed.ResultsSignificant differences in CD34 ⁺ cell yield among different age groups were observed. Furthermore, higher CD34 ⁺ cell yields were obtained in patients receiving chemotherapy as part of the mobilization regimen than those without chemotherapy. A correlation was noted between the CD34 ⁺ cell yield and blood surrogate markers, including white blood cell count, absolute neutrophil count and pre-apheresis peripheral blood CD34 ⁺ cell count. Cut-off values of > 35 CD34 ⁺ cells/μL in patients < 15 years old and > 45 CD34 ⁺ cells/μL in patients ≥ 15 years old were strong predictors of an adequate PBSC collection in one apheresis session. For clinical use, ROC curves and tables were generated to assist advance planning for PBSC collection.ConclusionsThe pre-apheresis peripheral blood CD34 ⁺ cell count is most useful in predicting PBSC yield. Our new cut-off values have better operating characteristics for children than the conventional value of 20 CD34 ⁺ cells/μL used for adults.