Characterization of human embryonic stem cell-derived hematopoietic progenitor phenotype

Differentiation of human embryonic stem cells (hESCs) into hematopoietic lineages using various methods has been reported. However, the phenotype that precisely defines the hematopoietic progenitor compartment with clonogenic activities has yet to be determined. Here, we measured and characterized progenitor function of subfractions of cells prospectively isolated from human embryoid bodies (hEBs) during hematopoietic differentiation basing on surface markers CD45, CD34, CD43, and CD38. We report that hematopoietic progenitors predominantly resided in the CD45⁺ subset. CD43⁺ cells lacking CD45 expression were largely devoid of progenitor activity. However, progenitor activity and multipotentiality was more enriched in CD45⁺ cells co-expressing CD43. CD45⁺ subset co-expressing CD34 but lacking CD38 expression (CD45⁺CD34⁺CD38^-) were further enriched for CFU capacity compared to the CD45⁺CD34⁺CD38⁺ subset. Our study demonstrates a role of CD43 in enriching hematopoietic progenitors derived from hEBs and reveals a hierarchical organization of hESC-derived hematopoietic progenitor compartments defined by phenotypic markers.     

Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor

Background aimsMesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1α (SDF-1α) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro.MethodsWe monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor.ResultsCo-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34⁺ cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations—and conversely their hematopoiesis supportive functions—may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor.ConclusionsThese results support the notion that the CXCR4/SDF-1α axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.     

具有维持造血干/祖细胞能力的新型豆类凝集素的分离、纯化及功能鉴定

一种分离自豆类的新型凝集素不仅具有凝集活性,还具有体外长期维持造血干/祖细胞的能力.由眉豆（Dolichos lablab）中分离得到了这种多亚基的凝集素——FRIL（Flt3 receptor-interacting lectin）,并对它进行了核酸和蛋白质序列分析.免疫细胞分析显示,它的受体是CD34⁺造血干/祖细胞所特有的.在培养基中添加这种凝集素可长期维持CD34⁺细胞存活和增殖能力.以Flt3配基（FL）作为对照,在28天的培养时间内,相对于FL,FRIL可维持细胞较高的G0/G1期比例（80%以上G0/G1期）和长期培养中（14天以上）1.5倍以上的集落形成量.可见FRIL通过滞留造血干/祖细胞于G0/G1期而维持它们的自我更新潜能.     

Circulating CD34+ hematopoietic progenitors in the harvesting peripheral blood stem cells; enhancement by recombinant human granulocyte colony-stimulating factor

The number of circulating progenitor cells increases during the period of hematopoietic recovery following myeloablative therapy. These progenitor cells were used for autologous transplantation in order to reconstitute hematopoiesis. As an indicator of the circulating progenitor cells, the number of granulocyte-macrophage colony forming units (CFU-GM), which is measured by means of a long-term cell culture, has been widely used. Recently, a cell surface marker, CD34, which can easily be measured by means of flowcytometry, was found to represent immature hematopoietic progenitor cells, which are very close to stem cells. Therefore, the relationship between the number of CD34 positive cells (CD34⁺ cells) and the number of CFU-GM in the peripheral blood following chemotherapy was studied in 9 patients selected to undergo autotransplantation. The number of peripheral blood CD34+ cells was found to be significantly correlated with that of CFU-GM (r = 0.81). When four out of 9 patients received recombinant human granulocyte-colony stimulating factor (rG-CSF) administration, a significant increase in the release of peripheral blood CD34⁺ cells as well as peripheral blood CFU-GM was observed (P<0.01). Thus, the measurement of CD34⁺ cells is useful for predicting the number of circulating CFU-GM.     

Testosterone influences renal electrolyte excretion in SHR/y and WKY males

Background

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH⁺ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH⁺ cellular subsets in bone marrow from pure red cell aplasia patients.

Results

In normal bone marrow, we identified three populations of cells, namely ALDH⁺CD34⁺, ALDH^-CD34⁺ and ALDH⁺CD34^- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH^-CD34⁺ cells, ALDH⁺CD34⁺ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH⁺CD34^- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH⁺CD34^- cells showed a CD71^bright, CD105⁺, CD45^- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH⁺CD34^- precursors were not detectable in patients with pure red cell aplasia (PRCA).

Conclusion

Our study, comparing surface antigen expression of ALDH⁺/CD34⁺, ALDH^-/CD34⁺ and ALDH⁺/CD34^- progenitor cell subsets in human bone marrow, clearly indicated that ALDH⁺CD34^- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.     

Ex vivo expansion of human umbilical cord blood hematopoietic progenitor cells in a novel three-dimensional culture system

A novel three-dimensional culture system for the ex vivo expansion of human umbilical cord blood (CB) hematopietic progenitor cells (HPCs) was developed by growing CB mononuclear cells on highly porous CultiSpher G microspheres coated with human bone marrow stromal cells in stirred flasks in the presence of supplemented cytokines. After 12 days, the number of total viable cells, colony-forming units in culture (CFU-C) and CD34⁺ cells present in the cultures reflected average increases of 7.7, 23.3 and 9.6-fold, respectively, and marked hematopoietic islands were formed on the surface of CultiSpher G.     

Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients

Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34⁺ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34⁺ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34⁺ cells as well as viability and clonogenic assays of CD34⁺ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34⁺ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34⁺ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.     

Expression of early hematopoietic markers in cord and mobilized blood

G-CSF mobilized peripheral blood and cord blood are major sources of hematopoietic progenitor cells. These cells are characterized by expression of “early” antigens. We have evaluated the coexpression of hematopoietic markers CD34, CD133, CD90, CDCP1, and CD117 and activation antigen CD38 using multicolor flow cytometry. We showed that cells positive for each particular antigen generate a separate population. The percentage of cells expressing each particular “early” antigen is twice as high in the cord blood as in the mobilized blood. The cell number with complex progenitor phenotype (CD34⁺/CD38^?/CD117^?, CD133⁺/CD34⁺/CD38^?, CDCP1⁺/CD34⁺/CD38^?, etc.) is equal in mobilized and cord blood. There is a strong positive correlation between CD34, CD133, CD117, and CDCP1 expression in both groups. Positive correlation was observed between CD90 and CD34, CD133, CDCP1, and CD117 expression only in cord blood; it was not significant in mobilized blood. The analyses of early antigens’ coexpression with activation CD38 marker did not confirm the hypothesis of sequential activation and loss of expression of the aforementioned antigens. We assume that there is global regulation of CD34, CD133, CDCP1, and CD117 expression. Expression of CD38 may be reversibly suppressed during maturation of the hematopoietic cells, and CD117 may be expressed on not only myeloid cells.     

Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells support hematopoietic recovery of X-irradiated human CD34+ cells

AimsThe potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed.Main methodsPlacental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin.Key findingsThe total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^? cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction.SignificanceThe present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.     

Immunophenotyping in Myelodysplastic Syndromes Can Add Prognostic Information to Well-Established and New Clinical Scores

Background

myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic clonal disorders. So, prognostic variables are important to separate patients with a similar biology and clinical outcome. We compared the importance of risk stratification in primary MDS of IPSS and WPSS with the just described revision of IPSS (IPSS-R), and examined if variables obtained by bone marrow immunophenotyping could add prognostic information to any of the scores.

Methods

In this prospective study of 101 cases of primary MDS we compared the relation of patients’ overall survival with WHO types, IPSS, IPSS-R, WPSS and phenotypic abnormalities of hematopoietic precursors. We examined aberrancies in myelomonocytic precursors and CD34⁺ cells. Patients were censored when receiving chemotherapy or BM transplantation. Survival analysis was made by Cox regressions and stability of the models was examined by bootstrap resampling.

Results

median age: 64 years (15–93). WHO types: 2 cases of 5q- syndrome, 7 of RA, 64 of RCDM and 28 of RAEB. In the univariate Cox analysis, increasing risk category of all scores, degree of anemia, higher percentage of BM blasts, higher number of CD34⁺ cells and their myeloid fractions besides increasing number of phenotypic abnormalities detected were significantly associated with a shorter survival. In the multivariate analysis comparing the three scores, IPSS-R was the only independent risk factor. Comparing WPSS with phenotypic variables (CD34⁺/CD13⁺ cells, CD34⁺/CD13⁻ cells and “total alterations”) the score and “CD34⁺/CD13⁺ cells” remained in the model. When IPSS was tested together with these phenotypic variables, only “CD34⁺/CD13⁺ cells”, and “total alterations” remained in the model. Testing IPSS-R with the phenotypic variables studied, only the score and “CD34⁺/CD13⁺ cells” entered the model.

Conclusions

Immunophenotypic analysis of myelomonocytic progenitors provides additional prognostic information to all clinical scores studied. IPSS-R improved risk stratification in MDS compared to the former scores.     

Cross Talk with Hematopoietic Cells Regulates the Endothelial Progenitor Cell Differentiation of CD34 Positive Cells

Introduction

Despite the crucial role of endothelial progenitor cells (EPCs) in vascular regeneration, the specific interactions between EPCs and hematopoietic cells remain unclear.

Methods

In EPC colony forming assays, we first demonstrated that the formation of EPC colonies was drastically increased in the coculture of CD34⁺ and CD34⁻ cells, and determined the optimal concentrations of CD34⁺ cells and CD34⁻ cells for spindle-shaped EPC differentiation.

Results

Functionally, the coculture of CD34⁺ and CD34⁻ cells resulted in a significant enhancement of adhesion, tube formation, and migration capacity compared with culture of CD34⁺ cells alone. Furthermore, blood flow recovery and capillary formation were remarkably increased by the coculture of CD34⁺ and CD34⁻ cells in a murine hind-limb ischemia model. To elucidate further the role of hematopoietic cells in EPC differentiation, we isolated different populations of hematopoietic cells. T lymphocytes (CD3⁺) markedly accelerated the early EPC status of CD34⁺ cells, while macrophages (CD11b⁺) or megakaryocytes (CD41⁺) specifically promoted large EPC colonies.

Conclusion

Our results suggest that specific populations of hematopoietic cells play a role in the EPC differentiation of CD34⁺ cells, a finding that may aid in the development of a novel cell therapy strategy to overcome the quantitative and qualitative limitations of EPC therapy.     

Retronectin enhances lentivirus-mediated gene delivery into hematopoietic progenitor cells

Genetic modification of hematopoietic stem cells holds great promise in the treatment of hematopoietic disorders. However, clinical application of gene delivery has been limited, in part, by low gene transfer efficiency. To overcome this problem, we investigated the effect of retronectin (RN) on lentiviral-mediated gene delivery into hematopoietic progenitor cells (HPCs) derived from bone marrow both in vitro and in vivo. RN has been shown to enhance transduction by promoting colocalization of lentivirus and target cells. We found that RN enhanced lentiviral transfer of the VENUS transgene into cultured c-Kit⁺ Lin^? HPCs. As a complementary approach, in vivo gene delivery was performed by subjecting mice to intra-bone marrow injection of lentivirus or a mixture of RN and lentivirus. We found that co-injection with RN increased the number of VENUS-expressing c-Kit⁺ Lin^? HPCs in bone marrow by 2-fold. Further analysis of VENUS expression in colony-forming cells from the bone marrow of these animals revealed that RN increased gene delivery among these cells by 4-fold. In conclusion, RN is effective in enhancing lentivirus-mediated gene delivery into HPCs.     

Characterizing the Lymphopoietic Kinetics and Features of Hematopoietic Progenitors Contained in the Adult Murine Liver In Vivo

The appearance of donor-derived lymphocytes in liver transplant patients suggests that adult livers may contain cells capable of lymphopoiesis. However, only a few published studies have addressed the lymphopoietic capacity of adult liver cells, and its kinetics and features remain unclear. Herein, we investigated the lymphopoietic capacity of adult liver mononuclear cells (MNCs) and purified liver hematopoietic progenitor cells (HPCs) in vivo. Similar to bone-marrow transplantation (BMT), transplantation of liver MNCs alone was able to rescue survival of lethally irradiated mice. In terms of kinetics, liver MNC-derived myeloid lineage cells reconstituted more slowly than those from BMT. Liver MNC-derived lymphocyte lineage cells in the blood, spleen and BM also reconstituted more slowly than BMT, but lymphocytes in the liver recovered at a similar rate. Interestingly, liver MNCs predominantly gave rise to CD3⁺CD19⁻ T cells in both irradiated WT and non-irradiated lymphocyte-deficient Rag-1^−/−Il2rg^−/− recipients. To define the lymphopoietic potential of various cell populations within liver MNCs, we transplanted purified lineage-negative (Lin⁻) liver HPCs into recipient mice. Unlike total liver MNCs, liver HPCs reconstituted T and B cells in similar frequencies to BMT. We further determined that the predominance of T cells observed after transplanting total liver MNCs likely originated from mature T cells, as purified donor liver T cells proliferated in the recipients and gave rise to CD8⁺ T cells. Thus, the capacity of donor adult liver cells to reconstitute lymphocytes in recipients derives from both HPCs and mature T cells contained in the liver MNC population.     

Direct observation of hematopoietic progenitor chimerism in fetal freemartin cattle

Background

Cattle twins are well known as blood chimeras. However, chimerism in the actual hematopoietic progenitor compartment has not been directly investigated. Here, we analyzed fetal liver of chimeric freemartin cattle by combining a new anti-bovine CD34 antibody and Y-chromosome specific in situ hybridization.

Results

Bull-derived CD34⁺ cells were detected in the liver of the female sibling (freemartin) at 60 days gestation. The level of bull-derived CD34⁺ cells was lower in the freemartin than in its male siblings. Bull (Y⁺) and cow hematopoietic cells often occurred in separate clusters. Around clusters of Y⁺CD34⁺ cells, Y⁺CD34^- cells were typically observed. The thymi were also strongly chimeric at 60 days of gestation.

Conclusion

The fetal freemartin liver contains clusters of bull-derived hematopoietic progenitors, suggesting clonal expansion and differentiation. Even the roots of the hematopoietic system in cattle twins are thus strongly chimeric from the early stages of fetal development. However, the hematopoietic seeding of fetal liver apparently started already before the onset of functional vascular anastomosis.     

Defining “poor mobilizer” in pediatric patients who need an autologous peripheral blood progenitor cell transplantation

The definition of poor mobilizers is not clear in pediatric patients undergoing autologous peripheral blood hematopoietic progenitor cell (HPC) mobilization. Most studies conducted in children define those variables related to the collection of HPC after leukapheresis, but the information regarding exclusively the mobilization process is scarce. In our experience, most children (92.2%) reach the target CD34⁺ cell dose for autologous peripheral blood progenitor cell transplantation if CD34⁺ cell count was higher than 10/μL. No differences were observed between those with >20 CD34⁺ cells/μL and 11–20 CD34⁺ cells/μL. In this study, we analyzed the variables that influence CD34⁺ cell count; we found that prior use of radiotherapy was the main variable related to poor mobilization. Patients diagnosed with Ewing sarcoma, treated with radiotherapy and mobilized with standard doses of granulocyte colony-stimulating factor (G-CSF) were also at a high risk of mobilization failure. In these patients, we should consider mobilization with high dose G-CSF and be prepared with new mobilization agents to avoid delay on their course of chemotherapy.     

In vitro T lymphopoiesis: A model system for stem cell gene therapy for AIDS

Abstract: Stable introduction of therapeutic genes into hematopoietic stem cells has the potential to reconstitute immunity in individuals with HIV infection. However, many important questions regarding the safety and efficacy of this approach remain unanswered and may be addressed in a non-human primate model. To facilitate evaluation of expression of foreign genes in T cells derived from transduced hematopoietic progenitor cells, we have established a culture system that supports the differentiation of rhesus macaque and human CD34⁺ bone marrow derived cells into mature T cells. Thymic stromal monolayers were prepared from the adherent cell fraction of collagenase digested fetal or neonatal thymus. After 10–14 days, purified rhesus CD34⁺ bone marrow-derived cells cultured on thymic stromal monolayers yielded CD3⁺CD4⁺CD8⁺, CD3⁺CD4⁺CD8^?, and CD3⁺CD4^?CD8⁺ cells. Following stimulation with mitogens, these T cells derived from CD34⁺ cells could be expanded over 1,000-fold and maintained in culture for up to 20 weeks. We next evaluated the ability of rhesus CD34⁺ cells transduced with a retroviral vector containing the marker gene neo to undergo in vitro T cell differentiation. CD34⁺ cells transduced in the presence of bone marrow stroma and then cultured on rhesus thymic stroma resulted in T cells containing the retroviral marker gene. These studies should facilitate both in vitro and in vivo studies of hematopoietic stem cell therapeutic strategies for AIDS.     

Circulating Progenitor Cells and Vascular Dysfunction in Chronic Obstructive Pulmonary Disease

Background

In chronic obstructive pulmonary disease (COPD), decreased progenitor cells and impairment of systemic vascular function have been suggested to confer higher cardiovascular risk. The origin of these changes and their relationship with alterations in the pulmonary circulation are unknown.

Objectives

To investigate whether changes in the number of circulating hematopoietic progenitor cells are associated with pulmonary hypertension or changes in endothelial function.

Methods

62 COPD patients and 35 controls (18 non-smokers and 17 smokers) without cardiovascular risk factors other than cigarette smoking were studied. The number of circulating progenitors was measured as CD45⁺CD34⁺CD133⁺ labeled cells by flow cytometry. Endothelial function was assessed by flow-mediated dilation. Markers of inflammation and angiogenesis were also measured in all subjects.

Results

Compared with controls, the number of circulating progenitor cells was reduced in COPD patients. Progenitor cells did not differ between control smokers and non-smokers. COPD patients with pulmonary hypertension showed greater number of progenitor cells than those without pulmonary hypertension. Systemic endothelial function was worse in both control smokers and COPD patients. Interleukin-6, fibrinogen, high sensitivity C-reactive protein, vascular endothelial growth factor and tumor necrosis factor were increased in COPD. In COPD patients, the number of circulating progenitor cells was inversely related to the flow-mediated dilation of systemic arteries.

Conclusions

Pulmonary and systemic vascular impairment in COPD is associated with cigarette smoking but not with the reduced number of circulating hematopoietic progenitors. The latter appears to be a consequence of the disease itself not related to smoking habit.     

Meeting the COVID challenge: Optimizing vCD34+ in cryopreserved HPC samples for implementation of an external QA Program

BackgroundThe COVID-19 pandemic has forced a fundamental change in the global procurement of allogeneic hematopoietic progenitor cells (HPCs) for transplantation. To better meet the emergent challenges of transporting cryopreserved allogeneic HPC during pandemics, there is an urgent need for External Quality Assurance (EQA) programs to evaluate reproducibility and harmonization of viable CD34⁺ cell (vCD34⁺) HPC enumeration, as the current EQA programs are unsuitable for analysis of vCD34⁺. The cost-effective distribution of HPC cryopreserved reference samples (CRSs) with acceptable reproducibility and specificity is key to the success of a vCD34⁺ EQA program.MethodsCryopreserved HPC samples (n = 11) were either stored on dry ice for 1 to 4 days or for 1 day followed by liquid nitrogen (LN) storage for 1 to 3 days to assess optimal conditions for vCD34⁺ EQA. Flow cytometric enumeration of vCD34⁺ HPCs was performed using a single platform assay combined with 7-AAD viability dye exclusion. The optimum transportation condition was validated in pilot and multicenter national studies (n = 12).ResultsA combination of 1 day on dry ice followed by LN storage stabilized viability compared with continuous storage on dry ice. This study demonstrates that dispatch of CRSs on dry ice to recipient centers across a distance of ≤4000 km within 26 h, followed by LN storage, resulted in reproducible intercenter vCD34⁺ enumeration. The estimated cost of safer and more convenient dry ice delivery is >20-fold lower than that of LN.ConclusionThis approach can form the basis for economically and scientifically acceptable distribution of CRSs for external vCD34⁺ EQA.