Differences between peripheral blood and cord blood in the kinetics of lineage-restricted hematopoietic cells: implications for delayed platelet recovery following cord blood transplantation

Cord blood (CB) cells are a useful source of hematopoietic cells for transplantation. The hematopoietic activities of CB cells are different from those of bone marrow and peripheral blood (PB) cells. Platelet recovery is significantly slower after transplantation with CB cells than with cells from other sources. However, the cellular mechanisms underlying these differences have not been elucidated. We compared the surface marker expression profiles of PB and CB hematopoietic cells. We focused on two surface markers of hematopoietic cell immaturity, i.e., CD34 and AC133. In addition to differences in surface marker expression, the PB and CB cells showed nonidentical differentiation pathways from AC133(+)CD34(+) (immature) hematopoietic cells to terminally differentiated cells. The majority of the AC133(+)CD34(+) PB cells initially lost AC133 expression and eventually became AC133(-)CD34(-) cells. In contrast, the AC133(+)CD34(+) CB cells did not go through the intermediate AC133(-)CD34(+) stage and lost both markers simultaneously. Meanwhile, the vast majority of megakaryocyte progenitors were of the AC133(-)CD34(+) phenotype. We conclude that the delayed recovery of platelets after CB transplantation is due to both subpopulation distribution and the process of differentiation from AC133(+)CD34(+) cells.     

Cord blood CD34+ cells cultured with FLT3L, stem cell factor, interleukin-6, and IL-3 produce CD11c+CD1a-/c- myeloid dendritic cells

Methods that allow expansion of myeloid dendritic cells (MDCs) from CD34(+) cells are potentially important for boosting anti-leukemic responses after cord blood (CB) hematopoietic stem cell transplantation (HSCT). We showed that the combination of early-acting cytokines FLT3-ligand (FL), stem cell factor (SCF), interleukin (IL)-3, and IL-6 supported the generation of CD11c(+)CD16() CD1a()/c() MDCs from CB CD34(+) cells or CB myeloid precursors. Early-acting cytokine-derived MDCs were maintained within the myeloid CD33(+)CD14()CD15() precursors with a mean of 4 x 10(6) cells generated from 1-4 x 10(4) CB CD34(+) cells or myeloid precursors after 2 weeks. After 8-12 days of culture the MDCs expressed higher levels of HLA-DR antigen but lower levels of CD40 and CD86 antigen, compared to adult blood MDCs. At this stage of differentiation, the early-acting cytokine-derived MDCs had acquired the ability to induce greater allogeneic T cell proliferation than monocytes or granulocytes derived from same culture. Early-acting cytokine-derived MDCs exposed to the cytokine cocktail (CC) comprising IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and prostaglandin E (PGE)-2, upregulated the surface co-stimulatory molecules CD40 and CD86 and enhanced allogeneic T cell proliferation, as is characteristic of MDCs maturation. The reliable production of MDCs from CB CD34(+) cells provides a novel way to study their lineage commitment pathway(s) and also a potential means of enriching CB with MDCs to improve prospects for DC immunotherapy following CB HSCT.     

Clonal heterogeneity in growth kinetics of CD34+CD38- human cord blood cells in vitro is correlated with gene expression pattern and telomere length

Human hematopoietic stem cells (HSCs) are characterized by an extensive proliferative capacity that decreases from fetal liver to cord blood (CB) to adult bone marrow. In previous studies, it was demonstrated that the proliferative capacity of individual CD34+CD38- HSC clones is correlated with their growth kinetics in vitro and that HSC turnover in vivo can be estimated by telomere-length measurements.The present study was aimed at the characterization of the clonal composition of CD34+CD38- human umbilical CB cells in terms of growth kinetics, telomere length, and gene expression profile. For this purpose, individual CD34+CD38- CB cells were sorted into 96-well plates containing serum-free medium supplemented with six growth factors. During expansion, cell numbers in each individual well were scored in 3-day intervals. Once sufficient cell numbers were achieved, telomere length was measured by flow fluorescence in situ hybridization (flow FISH). In a second set of experiments, gene expression and colony-forming capacity were analyzed in slowly growing clones as compared with fast-growing clones, using linear amplification and oligonucleotide microarrays (HG-U133A; Affymetrix).Individual CD34+CD38- cells from CB displayed an extensive functional heterogeneity in growth kinetics. Among highly proliferative clones, the most slowly growing clones were characterized by the longest telomeres. Furthermore, significant differences in gene expression were detected between slow- and fast-growing clones, whereas no significant difference in colony-forming capacity was observed. These data provide further evidence for a functional hierarchy in the human HSC compartment and suggest a link between telomere length and proliferation capacity of individual HSC clones.     

Characterization of serum-free ex vivo-expanded hematopoietic stem cells derived from human umbilical cord blood CD133(+) cells

The development of ex vivo expansion of hematopoietic stem cells (HSCs) is a promising approach to restore the required bone marrow function of patients with hematological disorders. Previously, we have reported the development of an optimized serum-free and cytokines-limited defined medium using statistic methodology for umbilical cord blood-derived HSC expansion. The aim of this study was to analyze further the characteristics and functions of cells in vitro and in vivo when cultured in this defined medium. After a 7-day batch culture, the average absolute fold expansions for CD133(+) cells, CD34(+)CD133(+) cells, CD34(+)CD38() cells, CD133(+)CD38(-) cells, CD34(+)CXCR4(+) cells, CD133(+)CXCR4(+) cells, and long-term culture-initiating cells were 21-, 20-, 723-, 618-, 160-, 384-, and 8-fold, respectively. The high enrichment of CD38(-) cells and CXCR4(+) cells of the CD34(+) subpopulation provided a very early uncommitted HSC proliferation and homing ability. Furthermore, the expanded cells showed a high level of telomerase activity to maintain their telomere length and repopulated the lethally irradiated NOD/SCID mice in vivo. These results indicated that the cytokines limited expanded cells from CD133(+) cells could substantially support simultaneous expansion of various stem/progenitor cells and engraft with the expanded cells from a low number of HSCs initially.     

Immunophenotype and functional characteristics of human primitive CD34-negative hematopoietic stem cells: the significance of the intra-bone marrow injection

The biology of hematopoietic stem cell (HSC) is a current topic of interest which has important implications for clinical HSC transplantation as well as for the basic research of HSC. The most primitive HSCs in mammals, including mice and humans, have long been believed to be CD34 antigen (Ag)-positive (CD34(+)) cells. In fact, bone marrow (BM), peripheral blood (PB), and cord blood (CB) stem cell transplantation studies indicate that a CD34(+) subpopulation in the BM, PB, or CB can provide durable long-term donor-derived lymphohematopoietic reconstitution. Therefore, CD34 Ag was used to identify/purify immature HSCs. However, Osawa et al. reported that murine long-term lymphohematopoietic reconstituting HSCs are lineage marker-negative (Lin(-)) c-kit(+)Sca-1(+)CD34-low/negative (CD34(low/-)), which are called CD34(low/-) KSL cells. Recently, human CB-derived CD34(-) HSCs, a counterpart of murine CD34(low/-) KSL cells, were successfully identified using an intra-bone marrow injection (IBMI) method. This review will update the concept of the immunophenotype and the functional characteristics of human primitive CD34(-) HSCs. In addition, the significance of the application of the IBMI technique in clinical HSC transplantation is also discussed. Recent rapid advances in understanding the biological nature of HSCs may make it possible to fully characterize the most primitive class of human HSCs in the near future.     

Inhibition of CD26 in human cord blood CD34+ cells enhances their engraftment of nonobese diabetic/severe combined immunodeficiency mice

CD26, a surface serine dipeptidylpeptidase IV (DPPIV) expressed on different cell types, cleaves the amino-terminal dipeptide from some chemokines, including stromal-derived factor-1 (SDF-1/CXCL12). SDF-1/CXCL12 plays important roles in hematopoietic stem cell (HSC) homing, engraftment, and mobilization. Inhibition of CD26 peptidase activity enhances homing, engraftment, and competitive repopulation in congenic mouse bone marrow cell transplants. Our studies evaluated a role for CD26 in in vivo engraftment of HSCs from human umbilical cord blood (CB) into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Pretreating purified CD34(+) human CB cells with Diprotin A, a DPPIV inhibitor, for 15 min significantly enhanced engraftment. Treatment did not affect differentiation of CD34(+) cells in vivo, as measured phenotypically by human markers CD33, CD38, CD19, and CD34. We found that the percentage of CD26(+) cells within the more immature cells (CD34(+)CD38()) was significantly higher than in the more mature population (CD34(+)CD38(+)). These results suggest that inhibition of CD26 may be one way to enhance engraftment of limiting numbers of stem cells during CB transplantation.     

In vitro proliferation potential of AC133 positive cells in peripheral blood

AC133 antigen is a novel marker for human hematopoietic stem/progenitor cells. In this study, we examined the expression and proliferation potential of AC133(+) cells obtained from steady-state peripheral blood (PB). The proportion of AC133(+) cells in the CD34(+) subpopulation of steady-state PB was significantly lower than that of cord blood (CB), although that of cytokine-mobilized PB was higher than that of CB. The proliferation potential of AC133(+)CD34(+) and AC133(-)CD34(+) cells was examined by colony-forming analysis and analysis of long-term culture-initiating cells (LTC-IC). Although the total number of colony-forming cells was essentially the same in the AC133(+)CD34(+) fraction as in the AC133(-)CD34(+) fraction, the proportion of LTC-IC was much higher in the AC133(+)CD34(+) fraction. Virtually no LTC-IC were detected in the AC133(-)CD34(+) fraction. In addition, the features of the colonies grown from these two fractions were quite different. Approximately 70% of the colonies derived from the AC133(+)CD34(+) fraction were granulocyte-macrophage colonies, whereas more than 90% of the colonies derived from the AC133(-)CD34(+) fraction were erythroid colonies. Furthermore, an ex vivo expansion study observed expansion of colony-forming cells only in the AC133(+)CD34(+) population, and not in the AC133(-)CD34(+) population. These findings suggest that to isolate primitive hematopoietic cells from steady-state PB, selection by AC133 expression is better than selection by CD34 expression.     

Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity

The replacement of bone marrow (BM) as a conventional source of stem cell (SC) by umbilical cord blood (UCB) and granulocyte-colony stimulating factor-mobilized peripheral blood SC (PBSC) has brought about clinical advantages. However, several studies have demonstrated that UCB CD34(+) cells and PBSC significantly differ from BM CD34(+) cells qualitatively and quantitatively. Here, we quantified the number of SC in purified BM, UCB CD34(+) cells, and CD34(+) PBSC using in vitro and in vivo assays for human hematopoietic SC (HSC) activity. A cobblestone area-forming cell (CAFC) assay showed that UCB CD34(+) cells contained the highest frequency of CAFC(wk6) (3.6- to tenfold higher than BM CD34(+) cells and PBSC, respectively), and the engraftment capacity in vivo by nonobese diabetic/severe combined immunodeficiency repopulation assay was also significantly greater than BM CD34(+), with a higher proportion of CD45(+) cells detected in the recipients at a lower cell dose. To understand the molecular characteristics underlying these functional differences, we performed several DNA microarray experiments using Affymetrix gene chips, containing 12,600 genes. Comparative analysis of gene-expression profiles showed differential expression of 51 genes between BM and UCB CD34(+) SC and 64 genes between BM CD34(+) cells and PBSC. These genes are involved in proliferation, differentiation, apoptosis, and engraftment capacity of SC. Thus, the molecular expression profiles reported here confirmed functional differences observed among the SC sources. Moreover, this report provides new insights to describe the molecular phenotype of CD34(+) HSC and leads to a better understanding of the discrepancy among the SC sources.     

Combined characterization of microRNA and mRNA profiles delineates early differentiation pathways of CD133+ and CD34+ hematopoietic stem and progenitor cells

MicroRNAs (miRNAs) have been shown to play an important role in hematopoiesis. To elucidate the role of miRNAs in the early steps of hematopoiesis, we directly compared donor-matched CD133(+) cells with the more differentiated CD34(+) CD133(-) and CD34(-) CD133(-) cells from bone marrow on the miRNA and mRNA level. Using quantitative whole genome miRNA microarray and sequencing-based profiling, we found that between 109 (CD133(+) ) and 216 (CD34(-) CD133(-) ) miRNAs were expressed. Quantification revealed that the 25 highest expressed miRNAs accounted for 73% of the total miRNA pool. miR-142-3p was the highest expressed miRNA with up to 2,000 copies per cell in CD34(+) CD133(-) cells. Eighteen miRNAs were significantly differentially expressed between CD133(+) and CD34(+) CD133(-) cells. We analyzed their biological role by examining the coexpression of miRNAs and its bioinformatically predicted mRNA targets and luciferase-based reporter assays. We provide the first evidence for a direct regulation of CD133 by miR-142-3p as well as tropomyosin 1 and frizzled homolog 5 by miR-29a. Overexpression of miRNAs in CD133(+) cells demonstrated that miR-142-3p has a negative influence on the overall colony-forming ability. In conclusion, the miRNAs expressed differentially between the CD133(+) and CD34(+) CD133(-) cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling. These results are highly relevant for stem cell-based therapies with CD133(+) cells and delineate for the first time how the stem cell character of CD133(+) cells is defined by the expression of specific miRNAs.     

Mitochondrial DNA sequence heterogeneity of single CD34+ cells after nonmyeloablative allogeneic stem cell transplantation

We applied a single-cell method to detect mitochondrial DNA (mtDNA) mutations to evaluate the reconstitution of hematopoietic stem cells (HSCs) and committed progenitor cells after nonmyeloablative allogeneic stem cell transplantation in humans. In a total of 1,958 single CD34(+) cells from six human leukocyte antigen-matched sibling donor and recipient pairs, individual CD34(+) clones were recognized based on the observed donor- or recipient-specific mtDNA sequence somatic alteration. There was no overall reduction of mtDNA heterogeneity among CD34(+) cells from the recipient after transplantation. Samples collected from two donors over time showed the persistence of certain CD34(+) clones marked by specific mutations. Our results demonstrate the feasibility of distinguishing donor and recipient individual CD34(+) clones based on mtDNA mutations during engraftment. HSCs were not limited in number, and similar mtDNA heterogeneity levels suggested representation of the total stem cell compartment during rapid hematopoietic reconstitution in the recipient. Disclosure of potential conflicts of interest is found at the end of this article.     

Validation of a flow cytometry based chemokine internalization assay for use in evaluating the pharmacodynamic response to a receptor antagonist

Background

Mobilized-peripheral blood hematopoietic stem cells (HSCs) have been used for transplantation, immunotherapy, and cardiovascular regenerative medicine. Agents used for HSC mobilization include G-CSF and the CXCR4 inhibitor AMD3100 (plerixafor). The HSCs cells mobilized by each agent may contain different subtypes and have different functions. To characterize mobilized HSCs used for clinical applications, microRNA (miRNA) profiling and gene expression profiling were used to compare AMD3100-mobilized CD133+ cells from 4 subjects, AMD3100 plus G-CSF-mobilized CD133+ cells from 4 subjects and G-CSF-mobilized CD34+ cells from 5 subjects. The HSCs were compared to peripheral blood leukocytes (PBLs) from 7 subjects.

Results

Hierarchical clustering of miRNAs separated HSCs from PBLs. miRNAs up-regulated in all HSCs included hematopoiesis-associated miRNA; miR-126, miR-10a, miR-221 and miR-17-92 cluster. miRNAs up-regulated in PBLs included miR-142-3p, -218, -21, and -379. Hierarchical clustering analysis of miRNA expression separated the AMD3100-mobilized CD133+ cells from G-CSF-mobilized CD34+ cells. Gene expression analysis of the HSCs naturally segregated samples according to mobilization and isolation protocol and cell differentiation status.

Conclusion

HSCs and PBLs have unique miRNA and gene expression profiles. miRNA and gene expression microarrays maybe useful for assessing differences in HSCs.     

Positive selection and transplantation of autologous highly purified CD133(+) stem cells in resistant/relapsed chronic lymphocytic leukemia patients results in rapid hematopoietic reconstitution without an adequate leukemic cell purging.

We assessed the capacity of positively selected autologous CD133(+) hematopoietic stem cells (HSCs) to reconstitute lymphomyelopoiesis in chronic lymphocytic leukemia (CLL) patients receiving myeloablative chemotherapy. Ten resistant/relapsed CLL patients underwent HSC mobilization with chemotherapy and granulocyte-colony stimulating factor (G-CSF). Positive selection of circulating CD133(+) HSCs was performed by immunomagnetic technique. Highly purified HSCs were reinfused after busulphan/melphalan myeloablative treatment. A median number of 4.2 x 10(6) CD34(+) cells/kg and of 3.14 x 10(6) CD133(+) cells/kg were collected. Immunomagnetic selection resulted in the reinfusion of a median number of 2.45 x 10(6) CD133(+) cells/kg (median purity: 94.8%; median recovery: 84%) and 2.4 x 10(6) CD34(+) cells/kg (median purity: 93%; median recovery: 71%). HSC selection resulted in a median T cell and CD19(+)/CD5(+) cell depletion of 3.85 log and 2.8 log, respectively. At the molecular level, however, 7 of 8 valuable purified HSC fractions were contaminated by leukemic cells. All CLL patients showed rapid and sustained myeloid engraftment after reinfusion of purified CD133(+) cells. Immunologic reconstitution was comparable to that routinely observed in patients reinfused with unmanipulated leukapheresis products and no late infectious complications were observed. With a median follow-up of 28 months for transplanted patients, 5 patients are in clinical complete remission, 3 are in partial remission, and 1 is in progression. In conclusion, the reinfusion of highly purified CD133(+) HSCs allowed the rapid and sustained recovery of hematopoiesis after myeloablative treatment in resistant/relapsed CLL patients. However, the purging potential of positive selection of CD133(+) cells is not adequate to achieve tumor-free autografts.     

Cytokine expansion culture of cord blood CD34+ cells induces marked and sustained changes in adhesion receptor and CXCR4 expressions

Recent studies have demonstrated defective bone marrow homing of hematopoietic stem cells after cytokine expansion culture. Adhesion receptors (ARs) are essential to the homing process, and it is possible that cytokine culture modulates AR expression. We studied changes in expression of very late antigen-4 (VLA-4), VLA-5, L-selectin, leukocyte function-associated antigen-1 (LFA-1), CD44, and the stromal cell-derived factor-1 (SDF-1) receptor, CXCR4, during cytokine culture of cord blood (CB) CD34(+) cells. Expression of ARs was studied by flow cytometry on CB CD34(+) cells in whole blood, after purification and during culture for up to 10 days. Cells were cultured with stem cell factor (SCF), thrombopoietin (TPO), Flt3-ligand (Flt3), and G-CSF. Results showed that 80% or more of uncultured CD34(+) cells were positive for VLA-4, L-selectin, LFA-1, CD44, and CXCR4 while 50% were positive for VLA-5. Purification of CD34(+) cells did not affect AR expression, but cytokines increased expression three- to nine-fold throughout the 10-day culture period. In contrast, expression of CXCR4 decreased. Expression changes of ARs and CXCR4 on CD34(+)/CD38(-) cells mirrored those of the total CD34(+) population. The results indicate that cytokine culture significantly increases AR expression on CB CD34(+) cells, which may be related to the decrease in homing of cytokine-cultured hematopoietic stem cells.