Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE)--2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-platform ISHAGE)--4.9% (single-platform ISHAGE) in bone marrow. Our results show high correlation among all three methods. Since the three protocols are compatible, choosing the most convenient in terms of costs, simplicity and compliance with clinical results appears to be a logical consequence.     

Unraveling stem cell and progenitor subsets in autologous grafts according to methods of mobilization: implications for prediction of hematopoietic recovery

Background aimsIn the autologous setting, granulocyte colony-stimulating factor (G-CSF) (G), or, when failing, G plus plerixafor (G+P), are common regimens for mobilization of stem cells into peripheral blood. To delineate mobilization effects on graft composition and hematopoietic recovery, we compared contents of stem cells and progenitor cells in products of G+P- and G patients. Paired samples of G+P patients and prior insufficient G mobilization were available for analyses.MethodsSubset analyses of grafts were performed by flow cytometry and myeloid colony-forming assay. In search of new markers to ascertain graft quality, we determined the fractions of aldehyde dehydrogenase bright (ALDH^br) cells.ResultsG grafts contained higher percentages of CD34+ cells, CD34+CD38- cells, and committed progenitors (CD34+CD38+) compared with G+P grafts. A detailed characterization of the mobilized CD34+ cell subset showed higher percentages of CD38– among the CD34+ cells of the G+P group (P = 0.032). In contrast, the CD34+ cell subset in G grafts was characterized by a higher percentage of ALDH^br cells (P < 0.0001). Studying engraftment and day +100 graft function the G and G+P transplanted patients were comparable with respect to neutrophils, whereas in platelets they differed. In the prediction of engraftment and hematopoietic recovery, the dose of infused ALDH^br cells correlated best to both platelet (r = 0.565, P = 0.002) and neutrophil reconstitution (r = 0.366, P = 0.06).ConclusionsBesides showing dissimilar distributions of CD34+CD38– cells and progenitors in G and G+P grafts, this study further designated ALDH^br as a promising marker in determination and prediction of graft quality and hematopoietic recovery.     

Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N -succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34⁺CD38⁻ fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34⁺, CD133⁺ and CD38⁻) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC–MSC interaction might further enhance cord blood expansion on MSC.     

Peripheral blood and BM CD34+ CD38- cells show better resistance to cryopreservation than CD34+ CD38+ cells in autologous stem cell transplantation

BACKGROUND: We and others have shown a critical role for CD34+ CD38- cells in hematopoietic recovery after autologous stem cell transplantation (ASCT), in particular for platelet reconstitution. Thus a routine assessment of CD34+ CD38- cells in freezing-thawing procedures for autografting could represent an important tool for predicting poor engraftment. METHODS: To compare the impact of cryopreservation on CD34+ CD38+ and CD34+ CD38- hematopoietic stem cell subsets, 193 autograft products collected in 84 patients with malignancies were assessed before controlled-rate cryopreservation in 10% DMSO and after thawing for autografting. RESULTS: Cell counts after thawing were significantly different from the pre-freezing counts for total CD34+ (P<0.0001) and CD34+ CD38+ (P<0.0001) cells, but not for CD34+ CD38- cells (P=0.252). Median losses for CD34+, CD34+ CD38+ and CD34+ CD38- cells were, respectively, 11.8%, 11.4% and 0.0%. The magnitude of fresh/post-thawing percentage cell variation was significantly different when comparing between the CD34+ CD38+ and CD34+ CD38- cell subsets (P<0.001). Moreover, CD34+ CD38- cells exhibited recovery values > or =100% in 85/160 graft products, compared with 51/193 in CD34+ CD38+ cells (P<0.0001). Also, recovery values > or =90% were significantly better in the CD34+ CD38- (98/160 grafts) than in the CD34+ CD38+ subsets (89/193 grafts) (P<0.01). DISCUSSION: In this work we have demonstrated that CD34+ cells that do not express the CD38 Ag show a significantly better resistance to cryopreservation. This could represent another example of the particular ability of less committed progenitor cells to overcome environmental injuries. Moreover, we consider routine assessment of CD34+ CD38- cells before freezing as clinically relevant, but post-thawing controls may be avoided because of their good resistance to freezing.     

Ex vivo expansion of umbilical cord blood

The efficacy of cord blood (CB) transplantation is limited by the low cell dose available. Low cell doses at transplant are correlated with delayed engraftment, prolonged neutropenia and thrombocytopenia and elevated risk of graft failure. To potentially improve the efficacy of CB transplantation, approaches have been taken to increase the cell dose available. One approach is the transplantation of multiple cord units, another the use of ex vivo expansion. Evidence for a functional and phenotypic heterogeneity exists within the HSC population and one concern associated with ex vivo expansion is that the expansion of lower 'quality' hematopoietic progenitor cells (HPC) occurs at the expense of higher 'quality' HPC, thereby impacting the reserve of the graft. There is evidence that this is a valid concern while other evidence suggests that higher quality HPC are preserved and not exhausted. Currently, ex vivo expansion processes include: (1) liquid expansion: CD34+ or CD133+ cells are selected and cultured in medium containing factors targeting the proliferation and self-renewal of primitive hematopoietic progenitors; (2) co-culture expansion: unmanipulated CB cells are cultured with stromal components of the hematopoietic microenvironment, specifically mesenchymal stem cells (MSC), in medium containing growth factors; and (3) continuous perfusion: CB HPC are cultured with growth factors in 'bioreactors' rather than in static cultures. These approaches are discussed. Ultimately, the goal of ex vivo expansion is to increase the available dose of the CB cells responsible for successful engraftment, thereby reducing the time to engraftment and reducing the risk of graft failure.     

Fresh PBSC harvests, but not BM, show temperature-related loss of CD34 viability during storage and transport

BACKGROUND: The optimum conditions for storage and transport of freshly harvested HPC in the liquid state are uncertain. It is not specified in commonly applied standards for stem cell transplantation. We used a viable CD34 assay to determine the optimum temperature for maintaining progenitor cell viability in freshly harvested BM and PBSC. Our aim was to identify standardized conditions for storage and transport of marrow or peripheral blood products that would optimize CD34 recovery, leading to better transplant outcomes. METHODS: Samples were aseptically removed from 46 fresh HPC harvests (34 PBSC and 12 BM) and stored at refrigerated temperature (2-8 degrees C), room temperature (18-24 degrees C) and 37 degrees C for up to 72 h. Samples were analyzed for viable CD34+ cells/microL at 0, 24, 48 and 72 h. RESULTS: The mean viable CD34+ yield prior to storage was 7.7 x 10(6)/kg (range 0.7-30.3). The mean loss of viable CD34+ cells in HPC products at refrigerated temperature was 9.4%, 19.4% and 28% at 24, 48 and 72 h, respectively. In contrast, the mean loss of viable CD34+ cells at room temperature was 21.9%, 30.7% and 43.3% at 24, 48 and 72 h, respectively. No viable CD34+ cells remained after storage at 37 degrees C for 24 h. Only PBSC products and not BM showed temperature-related loss of CD34 viability. Greater loss of viable CD34+ cells was observed for allogeneic PBSC compared with autologous PBSC. DISCUSSION: These results demonstrate that the optimum temperature for maintaining the viability of CD34+ cells, during overnight storage and transport of freshly harvested HPC, is 2-8 degrees C. These findings will allow the development of standard guidelines for HPC storage and transport.     

Identification of primitive hematopoietic progenitor cells in the rhesus macaque

The close phylogenetic relationship of macaques to humans has resulted in their widespread use as a preclinical model for bone marrow transplantation and stem cell gene therapy. To facilitate further use of this model, we undertook analysis of hematopoietic cells using multiparametric flow cytometric analysis. Rhesus CD34+CD38- cells displayed a number of characteristics of primitive hematopoietic cells, including low forward and orthogonal scatter and the lack of expression of lineage-specific markers or human lymphocyte antigen-DR. Four-color flow cytometric analysis demonstrated that rhesus CD34+CD38- cells were heterogenous with respect to Thy-1 expression and were CD59dim. Quantitative limiting dilution long-term culture-initiating cell (LTC-IC) analysis demonstrated that CD34+CD38- cells were approximately 150-fold enriched for LTC-IC as compared with unfractionated bone marrow, and occurred at a frequency similar to that previously reported in humans. Thus, as in humans, the CD34+38- population of rhesus macaque bone marrow is enriched for primitive, multipotent hematopoietic progenitor cells.     

mRNA-mediated gene delivery into human progenitor cells promotes highly efficient protein expression

Gene transfer into human CD34+ haematopoietic progenitor cells (HPC) and multi-potent mesenchymal stromal cells (MSC) is an essential tool for numerous in vitro and in vivo applications including therapeutic strategies, such as tissue engineering and gene therapy. Virus based methods may be efficient, but bear risks like tumorigenesis and activation of immune responses. A safer alternative is non-viral gene transfer, which is considered to be less efficient and accomplished with high cell toxicity. The truncated low affinity nerve growth factor receptor (ALNGFR) is a marker gene approved for human in vivo application. Human CD34+ HPC and human MSC were transfected with in vitro-transcribed mRNA for DeltaLNGFR using the method of nucleofection. Transfection efficiency and cell viability were compared to plasmid-based nucleofection. Protein expression was assessed using flow cytometry over a time period of 10 days. Nucleofection of CD34+ HPC and MSC with mRNA resulted in significantly higher transfection efficiencies compared to plasmid transfection. Cell differentiation assays were performed after selecting DeltaLNGFR positive cells using a fluorescent activating cell sorter. Neither cell differentiation of MSC into chondrocytes, adipocytes and osteoblasts, nor differentiation of HPC into burst forming unit erythroid (BFU-E) colony forming unit-granulocyte, erythrocyte, macrophage and megakaryocyte (CFU-GEMM), and CFU-granulocyte-macrophage (GM) was reduced. mRNA based nucleofection is a powerful, highly efficient and non-toxic approach for transient labelling of human progenitor cells or, via transfection of selective proteins, for transient manipulation of stem cell function. It may be useful to transiently manipulate stem cell characteristics and thus combine principles of gene therapy and tissue engineering.     

Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood

BACKGROUND: Umbilical cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSC/HPC) for the reconstitution of the hematopoietic system after clinical transplantation. Cryopreservation of these cells is critical for UCB banking and transplantation as well as for research applications by providing readily available specimens. The objective of this study was to optimize cryopreservation conditions for CD34+ HSC/HPC from UCB. METHODS: Cryopreservation of CD34+ HSC/HPC from UCB after mononuclear cell (MNC) preparation was tested in a research-scale setup. Experimental variations were concentration of the cryoprotectant, the protein additive and cell concentration. In addition, protocols involving slow, serial addition and removal of DMSO were compared with standard protocols (fast addition and removal of DMSO) in order to avoid osmotic stress for the cryopreserved cells. Viability and recoveries of MNC, CD34+ cells and total colony-forming units (CFU) were calculated as read-outs. In addition, sterility testing of the collected UCB units before further processing was performed. RESULTS: The optimal conditions for cryopreservation of CD34+ HPC in MNC preparations were 10% DMSO and 2% human albumin at high cell concentrations (5 x 10(7) MNC/mL) with fast addition and removal of DMSO. After cryopreservation using a computer-controlled freezer, high viabilities (89%) and recoveries for CD34+ cells (89%) as well as for CFU (88%) were observed. Microbial contamination of the collected UCB samples was reduced to a rate of 6.4%. DISCUSSION: Optimized cryopreservation conditions were developed for UCB MNC in respect of the composition of the cryosolution. In addition, our results showed that fast addition of DMSO is essential for improved cryopreservation and post-thaw quality assessment results, whereas the speed of DMSO removal after thawing has little influence on the recoveries of CD34+ cells and CFU.     

Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis

Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.     

Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.     

Importance of CD34+ cell subsets in autologous PBSC transplantation: the mulhouse experience using CD34+CD38- cells as predictive tool for hematopoietic engraftment

Over the years, various biological parameters have been proposed for predicting rapidity and long term maintenance of hematopoietic engraftment after peripheral blood stem cell transplantation (PBSCT). Determination of the graft content in CFU-GM was the only one available until the end of the eighties. But, for technical reasons, and also because it does not actually evaluate the self-renewal potential of the cell products reinfused, it has now been commonly replaced by the determination of CD34+ cell amounts, which are known to contain the pluripotent hematopoietic stem cells. However, a frequent discrepancy still exists between the number of CD34+ cells reinfused and the engraftment efficiency. We have recently demonstrated a higher accuracy of the numbers of CD34+38- cells contained in graft products to predict rapidity of trilineage engraftment, which has further been confirmed by other investigators. Furthermore, we and others, have proposed a threshold dose of 5 x 10(4) CD34+38- cells/kg b.w. below which the trilineage engraftment kinetics are significantly slower and unpredictible. This "cut-off" value also appears to be a realistic clinical tool to decide if hematopoietic growth factor(s) must be administered or not after PBSCT. Indeed, when for example, rh-G-CSF administration after transplant of CD34+38- amounts < 5 x 10(4) kg has indisputable positive effects on the rapidity of neutrophil engraftment, length of hospitalization and posttransplant costs, enough to make it fully justified in this situation, it is absolutely not the case when it is administered after reinfusion of CD34+38- cell amounts > 5 x 10(4) /kg. In this case, posttransplant rh-G-CSF administration could even result in a decrease in stem cells with self-renewal potential of the graft, which should still raise more concerns for its indiscriminate and costly use.     

Single-cell image analysis to assess ABC-transporter-mediated efflux in highly purified hematopoietic progenitors

BACKGROUND: Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux. METHODS: We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter-mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively). RESULTS: The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein-mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 +/- 0.35 and 1.14 +/- 0.11, respectively; P = 0.01). P-glycoprotein-mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 +/- 0.19 and 1.28 +/- 0.18, respectively). CONCLUSION: The described method is a valuable tool for assessing ABC-transporter-mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells.     

Hematopoietic stem cells in research and clinical applications: The "CD34 issue"

In this paper, experimental findings concerning the kinetics of hematopoietic reconstitution are compared to corresponding clinical data. Although not clearly apparent, the transplantation practice seems to confirm the basic proposals of experimental hematology concerning hematopoietic reconstitution resulting from successive waves of repopulation stemming from different subpopulations of progenitor and stem cells. One of the "first rate" parameters in clinical transplantations in hematology; i.e. the CD34+ positive cell dose, has been discussed with respect to the functional heterogeneity and variability of cell populations endowed by expression of CD34. This parameter is useful only if the relative proportion of stem and progenitor cells in the CD34+ cell population is more or less maintained in a series of patients or donors. This proportion could vary with respect to the source, pathology, treatment, processing procedure, the graft ex vivo treatment and so on. Therefore, a universal dose of CD34+ cells cannot be defined. In addition, to avoid further confusion, the CD34+ cells should not be named "stem cells" or "progenitor cells" since these denominations only concern functionally characterized cell entities.     

A French single-center experience on allogeneic stem cell transplant cryopreservation during severe acute respiratory syndrome coronavirus 2 pandemic

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-SCT) is a curative treatment for chemo-resistant hematological malignancies. Because of transport restriction imposed by the coronavirus disease 2019 pandemic, regulatory bodies and societies recommended graft cryopreservation before recipient conditioning. However, the freezing and thawing processes, including washing steps, might impair CD34+ cell recovery and viability, thereby impacting the recipient engraftment. Over 1 year (between March 2020 and May 2021), we aimed to analyze the results of frozen/thawed peripheral blood stem cell allografts in terms of stem cell quality and clinical outcomes.MethodsTransplant quality was evaluated by comparing total nucleated cells (TNCs), CD34+ cells and colony-forming unit–granulocyte/macrophage (CFU-GM)/kg numbers as well as TNC and CD34+ cell viabilities before and after thawing. Intrinsic biological parameters such as granulocyte, platelet and CD34+ cell concentrations were analyzed, as they might be responsible for a quality loss. The impact of the CD34+ cell richness of the graft on TNC and CD34 yields was evaluated by designing three groups of transplants based on their CD34 /kg value at collection: >8 × 10 ⁶/kg, between 6 and 8 × 10⁶/kg and <6 × 10⁶/kg. The consequences of cryopreservation were compared in the fresh and thawed group by evaluating the main transplant outcomes.ResultsOver 1 year, 76 recipients were included in the study; 57 patients received a thawed and 19 patients a fresh allo-SCT. None received allo-SCT from a severe acute respiratory syndrome coronavirus 2–positive donor. The freezing of 57 transplants led to the storage of 309 bags, for a mean storage time (between freezing and thawing) of 14 days. For the fresh transplant group, only 41 bags were stored for potential future donor lymphocyte infusions. Regarding the graft characteristics at collection, median number of cryopreserved TNC and CD34+ cells/kg were greater than those for fresh infusions. After thawing, median yields were 74.0%, 69.0% and 48.0% for TNC, CD34+ cells and CFU-GM, respectively. The median TNC dose/kg obtained after thawing was 5.8 × 10⁸, with a median viability of 76%. The median CD34+ cells/kg was 5 × 10⁶, with a median viability of 87%. In the fresh transplant group, the median TNC/kg was 5.9 × 10⁸/kg, and the median CD34+ cells/kg and CFU-GM/kg were 6 × 10⁶/kg and 276.5 × 10⁴/kg, respectively. Sixty-one percent of the thawed transplants were out of specifications regarding the CD34+ cells/ kg requested cell dose (6 × 10⁶/kg) and 85% of them would have had this dose if their hematopoietic stem cell transplant had been infused fresh. Regarding fresh grafts, 15.8% contained less than 6 × 10⁶ CD34+ cells /kg and came from peripheral blood stem cells that did not reach 6 × 10⁶ CD34+ cells /kg at collection. Regarding the factor that impaired CD34 and TNC yield after thawing, no significant impact of the granulocyte count, the platelet count or the CD34+ cells concentration/µL was observed. However, grafts containing more than 8 × 10 ⁶/kg at collection showed a significantly lower TNC and CD34 yield.ConclusionsTransplant outcomes (engraftment, graft-versus-host disease, infections, relapse or death) were not significantly different between the two groups.     

Isolation of early hematopoietic cells, including megakaryocyte progenitors, in the ALDH-bright cell population of cryopreserved, banked UC blood

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors, whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed, washed, immunomagnetically depleted of cells expressing glycophorin A and CD14, reacted for flow cytometric detection of ALDH, and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H, 10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed, banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid, myeloid and megakaryocytic blood elements.     

Investigating the feasibility of stem cell enrichment mediated by immobilized selectins

Hematopoietic stem cell therapy is used to treat both malignant and non-malignant diseases, and enrichment of the hematopoietic stem and progenitor cells (HSPCs) has the potential to reduce the likelihood of graft vs host disease or relapse, potentially fatal complications associated with the therapy. Current commercial HSPC isolation technologies rely solely on the CD34 surface marker, and while they have proven to be invaluable, they can be time-consuming with variable recoveries reported. We propose that selectin-mediated enrichment could prove to be a quick and effective method for recovering HSPCs from adult bone marrow (ABM) on the basis of differences in rolling velocities and independently of CD34 expression. Purified CD34+ ABM cells and the unselected CD34- ABM cells were perfused over immobilized P-, E-, and L-selectin-IgG at physiologic wall shear stresses, and rolling velocities and cell retention data were collected. CD34+ ABM cells generally exhibited lower rolling velocities and higher retention than the unselected CD34- ABM cells on all three selectins. For initial CD34+ ABM cell concentrations ranging from 1% to 5%, we predict an increase in purity ranging from 5.2% to 36.1%, depending on the selectin used. Additionally, selectin-mediated cell enrichment is not limited to subsets of cells with inherent differences in rolling velocities. CD34+ KG1a cells and CD34- HL60 cells exhibited nearly identical rolling velocities on immobilized P-selectin-IgG over the entire range of shear stresses studied. However, when anti-CD34 antibody was co-immobilized with the P-selectin-IgG, the rolling velocity of the CD34+ KG1a cells was significantly reduced, making selectin-mediated cell enrichment a feasible option. Optimal cell enrichment in immobilized selectin surfaces can be achieved within 10 min, much faster than most current commercially available systems.     

Hemopoietic stem and precursor cell analysis in umbilical cord blood using the Sysmex SE-9000 IMI channel

Circulating hematopoietic progenitor cells (HPCs) are routinely measured by flow cytometry using CD34 expression. As an alternative, the "immature information" (IMI) channel measurement of the automated hematology analyzer Sysmex SE machines was developed. We tested the IMI channel HPC method with umbilical cord blood specimens. The IMI-HPCs were compared with CD34 counts and numbers of colony-forming units (CFUs). The IMI-HPC data were reproducible and dilution experiments yielded a log-linear relationship. The mean percentage of CD34(+) cells in 50 umbilical cords was 0.43 versus 0.11 of HPCs in the IMI channel (correlation coefficient r = 0.67). Absolute numbers yielded 96.79 x 10(6)/L CD34(+), 33.17 x 10(6)/L IMI-HPC, and 35.04 x 10(6)/L CFU-HPC. Receiver operating characteristics curves were made at various cutoff levels for CD34(+) cells to visualize sensitivity and specificity profiles. With median values of 13.56 x 10(6)/L for IMI-HPC and 20 x 10(6)/L for CD34(+) as cutoff points (the levels used in the laboratory to start stem cell pheresis), the percentage of false-negative observations was 70.4%. To exclude the influence of storage time, tests were repeated until 72 h after umbilical cord collection. Total white blood cell count decreased in most cases, whereas absolute number of IMI-HPC tended to increase in time. In conclusion, if HPC measurements in the IMI channel are used to monitor circulating stem cells during mobilization, one has to be aware of a very low correlation between these results and those of other methods such as CD34(+) analysis and colony growth. False-negative results do occur, but if events are seen in the IMI channel, this simple monitoring technique is useful to predict the presence of circulating stem cells.     

Autologous cell therapy as a new approach to treatment of radiation-induced bone marrow aplasia: preliminary study in a baboon model

The sparing of viable hematopoietic stem and progenitor cells located in underexposed bone marrow territories associated with the relative radioresistance of certain stem cell populations is the rationale for autologous cell therapy consisting of ex vivo expansion of residual cells after collection postirradiation. The feasibility of this treatment mainly depends on time constraints and hematopoietic cell threshold. We showed in this study that in the absence of early-acting mobilizing agent administration, subliminar amounts of CD34+ cells can be collected (1 x 10(6) CD34+ cells/100 mL bone marrow or for 1 L apheresis) from 6-Gy gamma globally irradiated baboons. Residual CD34+ cells were successfully expanded in serum-free medium in the presence of antiapoptotic cytokine combination (stem cell factor + FLT-3 ligand + thrombopoietin + interleukin 3, 50 ng/mL each, i.e., 4F): KCD34+ = x2.8 and x13.7 (n = 2). Moreover, we demonstrated the short-term neutrophil engraftment potential of a low-size mixed expanded graft (1.5 x 106 final CD34+cells/kg) issued from the coculture of unirradiated (20%) and 2.5-Gy in vitro irradiated (80%) CD34+ cells on an allogeneic stromal cell layer in the presence of 4F. Further preclinical research needs to be performed to clearly establish this therapeutic approach that could be optimized by the early administration of antiapoptotic cytokines.