Purified unfractionated G-CSF/chemotherapy mobilized CD34+ peripheral blood progenitors and not bone marrow CD34+ progenitors undergo selective erythroid differentiation in liquid culture in the presence of erythropoietin and stem cell factor

A combination of erythropoietin (EPO) plus stem cell factor (SCF) drove purified unfractionated granulocyte colony stimulating factor (G-CSF)/chemotherapy mobilized peripheral blood CD34⁺ cells to selective erythroid differentiation in liquid culture with an average 28-fold increase in the total cell number after 21 d. From day 6 of culture, cytologic and cytofluorimetric characterization revealed that cultured cells belonged to the erythroid lineage with a gradual wave of maturation along the erythroid pathway to terminal cells. A similar pattern of erythroid differentiation was observed when the same peripheral blood CD34⁺ cells were cultured with EPO plus SCF in serum-free medium. This cytokine combination produced selective erythroid differentiation with the complete exhaustion of the clonogenic potential on day 21. In parallel experiments the same circulating CD34⁺ cells underwent granulocytic/monocytic differentiation in liquid culture in response to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and SCF, demonstrating that these CD34⁺ progenitors had intact pluripotent differentiating potential. Conversely, bone marrow CD34⁺ cells isolated from bone marrow allografts were unable to selectively differentiate along the erythroid pathway when they were exposed to EPO plus SCF combination. However, these cells maintained a greater number of colony forming cells on day 21 of culture compared to mobilized peripheral blood CD34⁺ cells. This model is a simple and reliable way to obtain selective erythroid differentiation of peripheral blood G-CSF/chemotherapy mobilized CD34⁺ progenitor cells in liquid culture. The absence of cytokines such as GM-CSF and IL-3 in the culture medium permits studies on in vitro erythropoiesis without disturbance of prevalent myelopoiesis.     

CD133 allows elaborated discrimination and quantification of haematopoietic progenitor subsets in human haematopoietic stem cell transplants

The success of haematopoietic stem cell (HSC) transplantation largely depends on numbers of transplanted HSCs, which reside in the CD34⁺ populations of bone marrow (BM), peripheral blood stem cells (PBSC) and umbilical cord blood (UCB). More specifically HSCs reside in the CD38^low/? subpopulation, which cannot be objectively discriminated from mature CD34⁺ CD38⁺ progenitors. Thus, better marker combinations for the quantification of more primitive haematopoietic stem and progenitor cells in transplants are required. Recently, by combining CD34 and CD133 we could clearly distinguish CD133⁺ CD34⁺ multipotent and lympho‐myeloid from CD133^low CD34⁺ erythro‐myeloid progenitors in UCB samples. To qualify the assessment of CD133 for routine quality control of adult HSC sources, we analysed the developmental potentials of CD133⁺ and CD133^low subpopulations in BM and PBSC. Similar to UCB, CD133 expression objectively discriminated functionally distinct subpopulations in adult HSC sources. By implementing anti‐CD45RA staining, which separates multipotent (CD133⁺ CD34⁺ CD45RA^?) from lympho‐myeloid (CD133⁺ CD34⁺ CD45RA⁺) progenitor fractions, UCB was found to contain 2–3 times higher multipotent progenitor frequencies than BM and PBSC. To test for the consistency of CD133 expression, we compared CD133⁺ CD34⁺ contents of 128 UCB samples with maternal and obstetrical factors and obtained similar correlations to related studies focusing on CD34⁺ cell contents. In conclusion, implementation of anti‐CD133 staining into existing routine panels will improve the quality control analyses for HSC transplants.     

GROWTH FACTOR INDUCTION OF CYTOSOLIC PROTEIN TYROSINE KINASE ACTIVITY IN HUMAN HAEMOPOIETIC PROGENITOR CELLS ISOLATED BY FLOW CYTOMETRY

We employed a highly sensitive method to assay protein tyrosine kinase activity in extracts of subpopulations of CD34⁺ bone marrow progenitor cells isolated by fluorescence activated cell sorting in an attempt to better define how growth-factor induction of enzymatic activity relates to progenitor cell maturation. FACS analysis confirmed that, under the conditions employed, essentially all of the CD34⁺ cells in adult human marrow that lacked the CD38 antigen were devoid of the myeloid maturation marker CD33 as well as the lineage antigens: CD10, 13, 14, 15, 16, 19, 71 and glycophorin A. A variable portion (50–90%) of these CD34⁺, CD38^? progenitor cells expressed HLA-DR. CD34⁺, CD38^? cells that did not express HLA-DR were found to lack detectable levels of either membrane or cytosolic tyrosine kinase activity. HLA-DR⁺ progenitor cells that lacked CD38 possessed elevated levels of cytosolic tyrosine kinase activity but only low levels of plasma membrane activity. In contrast, CD34⁺ cells that expressed CD38 (and HLA-DR) possessed high levels of membrane-associated tyrosine kinase activity. A cocktail of haemopoietic growth factors that included IL-3, IL-6 and stem cell factor effectively induced tyrosine kinase activity in CD34⁺, CD38^?, HLA-DR^? progenitor cells. Growth factor induction of tyrosine kinase activity in these cells was not inhibited by actinomycin D or cyclohexamide. Most of the tyrosine kinase activity induced by these growth factors was recovered from the cytosolic fraction of disrupted cells. Thus, induction of cytosolic tyrosine kinase activity is an early event in the response of uncommitted haemopoietic cells to haemopoietic growth factors. Subsequent activation of membrane tyrosine kinases may initiate key transduction processes as these cells begin to differentiate.     

In vitro human embryonic stem cell hematopoiesis mimics MYB-independent yolk sac hematopoiesis

Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cell-initiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34⁺ hemogenic endothelial cells rounding up and developing into CD43⁺ hematopoietic cells without expression of MYB-eGFP. MYB-eGFP⁺ cells appeared relatively late in embryoid body cultures as CD34⁺CD43⁺CD45^−/lo cells. These MYB-eGFP⁺ cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb^−/− embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14⁺ macrophage cells were consistently eGFP⁻ and were derived from eGFP-precursors only. In summary, no evidence was obtained for in vitro generation of MYB⁺ hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage.     

Loss of quiescence and impaired function of CD34+/CD38low cells one year following autologous stem cell transplantation

Patients who have undergone autologous stem cell transplantation are subsequently more susceptible to chemotherapy-induced bone marrow toxicity. In the present study, bone marrow primitive progenitor cells were examined one year after autologous stem cell transplantation and compared with normal bone marrow and mobilized peripheral blood stem cells. Post-transplantation bone marrow contained a significantly lower percentage of quiescent cells in the CD34⁺/CD38^low fraction compared to normal bone marrow. In addition, we observed a strong decrease in stem cell/primitive progenitor frequency in post-transplantation CD34⁺ cells as defined by long-term culture assays. Measurement of the levels of reactive oxygen species by flow cytometry revealed comparable levels in post-transplantation and normal bone marrow CD34⁺/CD38^low cells, while significantly higher levels of reactive oxygen species were observed in CD34⁺/CD38^high cells following autologous stem cell transplantation compared to normal bone marrow. Moreover, post-transplantation CD34⁺ bone marrow cells demonstrated an increased sensitivity to buthionine sulfoximine, a trigger for endogenous production of reactive oxygen species. Gene expression analysis on CD34⁺ cells revealed a set of 195 genes, including HMOX1, EGR1, FOS and SIRPA that are persistently down-regulated in mobilized peripheral blood cells and post-transplantation bone marrow compared to normal bone marrow. In conclusion, our data indicate that the diminished regenerative capacity of bone marrow following autologous stem cell transplantation is possibly related to a loss of quiescence and a reduced tolerability to oxidative stress.     

Efficient retroviral transduction of human B-lymphoid and myeloid progenitors: marked inhibition of their growth by the <Emphasis Type="Italic">Pax5</Emphasis> transgene

We applied a coculture system for the genetic manipulation of human B-lymphoid and myeloid progenitor cells using murine bone marrow stromal cell support, and investigated the effects of forced Pax5 expression in both cell types. Cytokine-stimulated cord blood CD34⁺ cells could be transduced at 85% efficiency and 95% cell viability by a single 24-h infection with RD114-pseudotyped retroviral vectors, produced by the packaging cell line Plat-F and bicistronic vector plasmids pMXs-Ig, pMYs-Ig, or pMCs-Ig, encoding EGFP. Infected CD34⁺ cells were seeded onto HESS-5 cells in the presence of stem cell factor and granulocyte colony-stimulating factor, allowing the extensive production of B progenitors and granulocytic cells. We examined the cell number and CD34, CD33, CD19, and CD20 lambda and kappa expressions by flow cytometry. Ectopic expression of Pax5 in CD34⁺ cells resulted in small myeloid progenitors coexpressing CD33 and CD19 and inhibited myeloid differentiation. After 6 weeks, the number of Pax5-transduced CD19⁺ cells was 40-fold lower than that of control cells. However, the expression of CD20 and the κ/λ chain on Pax5-transduced CD19⁺ cells suggests that the Pax5 transgene may not interfere with their differentiation. This report is the first to describe the effects of forced Pax5 expression in human hematopoietic progenitors.     

The influence of extracellular matrix proteins and mesenchymal stem cells on erythropoietic cell maturation

Background and Objectives As part of the bone marrow niche, cellular and acellular components like mesenchymal stem cells (MSCs) and extracellular matrix (ECM) proteins influence human haematopoiesis. To identify factors able to improve the in vitro generation of red blood cells (RBCs), we investigated the effect of these factors on proliferation and differentiation of human haematopoietic stem cells (HSCs) into erythroid cells. Material and Methods Granulocyte colony‐stimulating factor–mobilized CD34⁺ HSCs were cultured for 16 days using an in vitro erythropoiesis assay as described previously (by our group). The HSCs were co‐cultured with MSCs in either direct or indirect contact and with different ECM proteins (fibronectin, laminin, collagen and a mixture of ECM proteins, called ECM gel). Results Co‐culturing of HSCs with ECM gel improved cell viability, and the presence of laminin slightly increased the maturation into enucleated RBCs. HSC expansion could not be improved by addition of any of the ECM proteins investigated. In contrast, fibronectin inhibited erythroid formation. Co‐culturing of HSCs with MSCs generally stimulated cell viability and HSC proliferation, however, in favour of the myeloid lineage. In summary, of all investigated factors, only laminin and ECM gel had a supportive effect on RBC development under the described in vitro culture conditions.     

Generation of multinuclear tartrate-resistant acid phosphatase positive osteoclasts in liquid culture of purified human peripheral blood CD34+ progenitors

Circulating CD34⁺ cells were isolated from leukapheresis products collected from patients with ovarian cancer. CD34^? contaminating cells, identified immediately after immunoselection, ranged from 5% to 25% in five different experiments and were predominantly CD3⁺ T-lymphocytes (range 2–12%), CD3^?/CD16⁺/CD56⁺ natural killer cells (range 2–11%) and rare mature CD15⁺/CD11b⁺ granulocytes (range 1–2%). CD34⁺ cells were cultured in liquid medium in the presence of interleukin-3, granulocyte-macrophage colony stimulating factor, stem cell factor, granulocyte colony stimulating factor and a powerful proliferation with prevalent differentiation along the granulocytic/monocytic lineage was obtained. After 10 d of culture a small but consistent number of early multinucleated osteoclasts were identified with a frequency of one cell per 700 granulocytic/monocytic cells, as revealed by cytologic examination. This observation was confirmed by staining for tartrate-resistant acid phosphatase activity which revealed red multinucleated elements with a frequency comparable to that reported above. Conversely, no osteoclasts were observed in those cultures in which macrophage overgrowth was obtained by culturing CD34⁺ cells until day 35. These observations suggest that circulating progenitors have a multilineage potential in vitro and contribute to the clarification of osteoclast development in humans; additionally, they provide the basis for the future development of optimized osteoclast culture techniques in liquid medium and the basic culture system, to test the distinct activity of 1,25(OH)₂D₃, parathyroid hormone, interleukin-11 and of other cytokines on osteoclast development in humans.     

Reconstitution of human haematopoiesis in non-obese diabetic/severe combined immunodeficient mice by clonal cells expanded from single CD34+CD38- cells expressing Flk2/Flt3

In the present study, we examined the expression of Flk2/Flt3, a tyrosine kinase receptor, on human cord blood CD34+ haematopoietic progenitor/stem cells. In flow cytometric analysis, Flk2/Flt3 was expressed on 80% of CD34+ cells and their immature subpopulations, CD34+CD33- and CD34+CD38- cells. Methycellulose clonal culture of sorted CD34+Flk2/Flt3+ and CD34+Flk2/Flt3- cells showed that most of myelocytic progenitors expressed Flk2/Flt3, but erythroid and haematopoietic multipotential progenitors were shared by both fractions. When 1 x 10(4) lineage marker-negative (Lin-)CD34+Flk2/Flt3- cells were transplanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, none of the recipients possessed human CD45+ cells in bone marrow 11-12 weeks after the transplantation. In contrast, all recipients transplanted with 1 x 10(4) Lin-CD34+Flk2/Flt3+ cells showed successful engraftment. Furthermore, clonal cells expanded from single Lin-CD34+CD38-Flk2/Flt3+ cells in the culture with Flk2/Flt3 ligand, stem cell factor, thrombopoietin, and a complex of interleukin 6/soluble interleukin 6 receptor were individually transplanted into NOD/SCID mice. At 20 to 21 weeks after the transplantation, three out of 10 clones harvested at d 7 of culture, and three out of six clones at d 14 could reconstitute human haematopoiesis in recipient marrow. These results demonstrated that Flk2/Flt3 was expressed on a wide variety of human haematopoietic cells including long-term-repopulating haematopoietic stem cells.     

Stem and progenitor cell systems in liver development and regeneration

The liver comprises two stem/progenitor cell systems: fetal and adult liver stem/progenitor cells. Fetal hepatic progenitor cells, derived from foregut endoderm, differentiate into mature hepatocytes and cholangiocytes during liver development. Adult hepatic progenitor cells contribute to regeneration after severe and chronic liver injuries. However, the characteristics of these somatic hepatic stem/progenitor cells remain unknown. Culture systems that can be used to analyze these cells were recently established and hepatic stem/progenitor cell‐specific surface markers including delta‐like 1 homolog (DLK), cluster of differentiation (CD) 13, CD133, and LIV2 were identified. Cells purified using antibodies against these markers proliferate for an extended period and differentiate into mature cells both in vitro and in vivo. Methods to force the differentiation of human embryonic stem and induced pluripotent stem (iPS) cells into hepatic progenitor cells have been recently established. We demonstrated that the CD13⁺CD133⁺ fraction of human iPS‐derived cells contained numerous hepatic progenitor‐like cells. These analyses of hepatic stem/progenitor cells derived from somatic tissues and pluripotent stem cells will contribute to the development of new therapies for severe liver diseases.     

Lack of DNA synthesis among CD34+ cells in cord blood and in cytokine-mobilized blood

Flow cytometric DNA analysis was performed in combination with three-colour immunological staining of cell surface antigens on density-separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell-cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5 μg per kg body weight of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Despite considerably increasing numbers of CD34⁺ PB MNC, the latter were not found to be in S/G₂M phase, whereas, among the CD34^? MNC, the proportion of cells in S/G₂M phase increased from <0.1% to 0.75 ± 0.4% (GM-CSF) and to 1.34 ± 0.75% (G-CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34⁺ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G₂M phase. The non-cycling status of PB and CB progenitor cells was confirmed by the analysis of CD34⁺ cells enriched from the two cell sources. However, in vitro stimulation of these progenitor cells using IL3, GM-CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34⁺ cells were in S/G₂M phase. The fact that cycling CD34⁺ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized ‘stem cells’ which may require the BM micro-environment for adequate proliferation in vivo     

Viable CD34+/CD133+ blood progenitor cell dose as a predictor of haematopoietic engraftment in multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation

Both CD34 (cluster of differentiation 34) and the more recently described CD133 are markers of primitive stem cells with haematopoietic repopulating ability. Most transplanting centres use a minimum number of CD34+ cells as the requirement for a transplant and consider this a predictor of haematopoietic engraftment. However, transplanted CD34+ cell dose does not always give a close correlation with time to engraftment nor explain delayed engraftment in some patients. We have retrospectively evaluated the potential of measuring viable CD133+ cell numbers in the autograft as an alternative predictor of haematological engraftment after autologous stem-cell transplantation in a cohort of patients with multiple myeloma (MM). We found an average 32% loss of viability of CD34+ cells in the post-thaw sample compared with the fresh sample. Of the original estimated CD34+ cell numbers transplanted per kg, 43% of the thawed samples were double positive for CD34+/CD133+. In this patient group, the CD34+/CD133+ subset gave the closest statistical correlation with time to neutrophil engraftment (p < 0.05), particularly for patients given above median (1.8 × 10⁶/kg) dose of the double-positive cells. The CD34+/CD133+ population was the only parameter to give a significant correlation with white cell engraftment in this patient cohort (p < 0.05). There was no significant correlation between CD34+, viable CD34+ or viable CD34+/CD133+ cells/kilogram with platelet engraftment. Determination of viable CD34+/CD133+ progenitor cell dose in the autograft may be a useful tool to predict neutrophil recovery after autologous transplantation than conventional assessment of CD34+ numbers. These results warrant further investigation of the role of CD133 in haematopoietic engraftment.     

Lectin-induced increase in clonogenic growth of haematopoietic progenitor cells

Abstract: The galactoside-specific plant lectin, Viscum album agglutinin (VAA-I) increases cellular parameters of natural host defence. It also binds to a variety of haematopoietic cells, including progenitors. We investigated whether VAA-I has a stimulatory effect on haematopoietic progenitor cells. Peripheral blood progenitor cells from 7 healthy volunteers were cultured in a colony assay with VAA-I plus erythropoietin (EPO) and stem cell factor (SCF). At 50 pg/ml VAA-I induced a significant increase in the cytokine-dependent clonogenic growth (52% in median, p<0.05). In another set of experiments purified CD34+ cells were isolated from the bone marrow aspirate of 4 patients with non-metastatic breast cancer using fluorescence-activated cell sorting. Binding to CD34+ cells was demonstrated by using directly fluorescence-conjugated VAA-I. Co-incubation with d -galactose significantly abrogated this effect. CD34+ cells were cultured in the presence of EPO, SCF, interleukin-3, granulocyte/monocyte colony-stimulating factor and granulocyte colony-stimulating factor. VAA-I alone had no measurable effect on the clonogenic growth of the isolated cells. However, at concentrations of 100 and 250 pg/ml VAA-I increased the cytokine-dependent proliferation and differentiation of CD34+ cells by a median of 75 and 85%, respectively. The results show that VAA-I binds to haematopoietic progenitor cells and has a co-stimulatory effect on their proliferation.     

The induction of immunogenic cell death by type II anti‐CD20 monoclonal antibodies has mechanistic differences compared with type I rituximab

The ASXL1 gene encodes a chromatin‐binding protein involved in epigenetic regulation in haematopoietic cells. Loss‐of‐function ASXL1 mutations occur in patients with a range of myeloid malignancies and are associated with adverse outcome. We have used lentiviral‐based shRNA technology to investigate the effects of ASXL1 silencing on cell proliferation, apoptosis, myeloid differentiation and global gene expression in human CD34⁺ cells differentiated along the myeloid lineage in vitro. ASXL1‐deficient cells showed a significant decrease in the generation of CD11b⁺ and CD15⁺ cells, implicating impaired granulomonocytic differentiation. Furthermore, colony‐forming assays showed a significant increase in the number of multipotent mixed lineage colony‐forming unit (CFU‐GEMM) colonies and a significant decrease in the numbers of granulocyte‐macrophage CFU (CFU‐GM) and granulocyte CFU (CFU‐G) colonies in ASXL1‐deficient cells. Our data suggests that ASXL1 knockdown perturbs human granulomonocytic differentiation. Gene expression profiling identified many deregulated genes in the ASXL1‐deficient cells differentiated along the granulomonocytic lineage, and pathway analysis showed that the most significantly deregulated pathway was the LXR/RXR activation pathway. ASXL1 may play a key role in recruiting the polycomb repressor complex 2 (PRC2) to specific loci, and we found over‐representation of PRC2 targets among the deregulated genes in ASXL1‐deficient cells. These findings shed light on the functional role of ASXL1 in human myeloid differentiation.     

Prolonged ex vivo culture of human bone marrow mesenchymal stem cells influences their supportive activity toward NOD/SCID-repopulating cells and committed progenitor cells of B lymphoid and myeloid lineages

Background

Bone marrow mesenchymal stem cells support proliferation and differentiation of hematopoietic progenitor cells in vitro. Since these cells constitute a rare subset of bone marrow cells, mesenchymal stem cell preparations for clinical purposes require a preparative step of ex vivo multiplication. The aim of our study was to analyze the influence of culture duration on mesenchymal stem cell supportive activity.

Design and Methods

Mesenchymal stem cells were expanded for up to ten passages. These cells and CD34+ cells were seeded in cytokine-free co-cultures after which the phenotype, clonogenic capacity and in vivo repopulating activity of harvested hematopoietic cells were assessed.

Results

Early passage mesenchymal stem cells supported hematopoietic progenitor cell expansion and differentiation toward both B lymphoid and myeloid lineages. Late passage mesenchymal stem cells did not support hematopoietic progenitor cell and myeloid cell outgrowth but maintained B-cell supportive ability. In vitro maintenance of NOD/SCID mouse repopulating cells cultured for 1 week in contact with mesenchymal stem cells was effective until the fourth passage of the mesenchymal cells and declined thereafter. The levels of engraftment of CD34⁺ cells in NOD/SCID mice was higher when these cells were co-injected with early passage mesenchymal stem cells; however mesenchymal cells expanded beyond nine passages were ineffective in promoting CD34⁺ cell engraftment. Non-contact cultures indicated that mesenchymal stem cell supportive activity involved diffusible factors. Among these, interleukins 6 and 8 contributed to the supportive activity of early passage mesenchymal stem cells but not to those of late passage cells. The phenotype, as well as fat, bone and cartilage differentiation capacity, of mesenchymal stem cells did not change during their culture.

Conclusions

Extended culture of mesenchymal stem cells alters the ability of these cells to support hematopoietic progenitor cells without causing concomitant changes in their phenotype or differentiation capacity.     

Cord blood-derived CD34+ hematopoietic cells with low mitochondrial mass are enriched in hematopoietic repopulating stem cell function

The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned balance between self-renewal, differentiation and proliferation. Recent studies have proposed that mitochondria regulate these processes. Although recent work has contributed to understanding the role of mitochondria during stem cell differentiation, it remains unclear whether the mitochondrial content/function affects human hematopoietic stem versus progenitor function. We found that mitochondrial mass correlates strongly with mitochondrial membrane potential in CD34⁺ hematopoietic stem/progenitor cells. We, therefore, sorted cord blood CD34⁺ cells on the basis of their mitochondrial mass and analyzed the in vitro homeostasis and clonogenic potential as well as the in vivo repopulating potential of CD34⁺ cells with high (CD34⁺ Mito^High) versus low (CD34⁺ Mito^Low) mitochondrial mass. The CD34⁺ Mito^Low fraction contained 6-fold more CD34⁺CD38⁻ primitive cells and was enriched in hematopoietic stem cell function, as demonstrated by its significantly greater hematopoietic reconstitution potential in immuno-deficient mice. In contrast, the CD34⁺ Mito^High fraction was more enriched in hematopoietic progenitor function with higher in vitro clonogenic capacity. In vitro differentiation of CD34⁺ Mito^Low cells was significantly delayed as compared to that of CD34⁺ Mito^High cells. The eventual complete differentiation of CD34⁺ Mito^Low cells, which coincided with a robust expansion of the CD34⁻ differentiated progeny, was accompanied by mitochondrial adaptation, as shown by significant increases in ATP production and expression of the mitochondrial genes ND1 and COX2. In conclusion, cord blood CD34⁺ cells with low levels of mitochondrial mass are enriched in hematopoietic repopulating stem cell function whereas high levels of mitochondrial mass identify hematopoietic progenitors. A mitochondrial response underlies hematopoietic stem/progenitor cell differentiation and proliferation of lineage-committed CD34⁻ cells.     

Lineage- and differentiation stage-specific expression of LSM-1 (LPAP), a possible substrate for CD45, in human hematopoietic cells

CD45, a transmembrane tyrosine phosphatase, is found on almost all nucleated hematopoietic cells and plays a crucial role in lymphocyte activation and differentiation. We recently achieved isolation of the human LSM-1 (hLSM-1) gene, whose product is a possible substrate for CD45, and we raised antibodies against the hLSM-1 protein. hLSM-1 expression in hematopoietic cells was examined with Northern and Western blot, fluorescence-activated cell sorter, and immunocytochemical analyses. It was found that in the lymphoid lineage, T and B lymphocytes as well as NK cells expressed LSM-1, whereas terminally differentiated plasma cells did not. As for the myeloid lineage, immature myeloid cells expressed LSM-1, whereas terminally differentiated granulocytes and monocytes did not. In the erythroid lineage, normal erythroblasts expressed very low levels of LSM-1, while erythroid cell lines (K562 and HEL) did not. Megakaryocytes did not express LSM-1. Both CD34⁺/CD33⁻ and CD34⁺/CD33⁺ hematopoietic progenitor cells weakly expressed LSM-1. These results showed that LSM-1 is expressed in a lineage- and differentiation stage-specific fashion. Am. J. Hematol. 54:1–11, 1997 © 1997 Wiley-Liss, Inc.     

Bone marrow characterization in sickle cell disease: inflammation and stress erythropoiesis lead to suboptimal CD34 recovery

Stress erythropoiesis and chronic inflammation in subjects with sickle cell disease (SCD) may have an impact on the bone marrow (BM) haematopoietic stem and progenitor cell (HSPC) quality and yield necessary for effective autologous, ex vivo HSPC gene therapy. BM from 19 subjects with SCD and five volunteers without SCD (non-SCD) was collected in different anticoagulants and processed immediately (day 0) or the following day (day 1). Inflammatory, contamination and aggregation markers within the mononuclear layer, and CD34, CD45 and Glycophorin-A (GPA) expression on HSPCs after CD34⁺ selection were analysed by conventional and imaging flow cytometry. Compared to non-SCD BM, multiple markers of inflammation, contamination (red cells, P < 0·01; platelets, P < 0·01) and aggregates (platelet/granulocytes, P < 0·01; mononuclear/red cells, P < 0·01) were higher in SCD BM. Total CD34⁺ cell count was lower in SCD BM (P < 0·05), however CD34⁺ count was higher in SCD BM when collected in acid citrate dextrose-A (ACDA) versus heparin (P < 0·05). Greater than 50% of CD34⁺ HSPCs from SCD BM are CD34^dim due to higher erythroid lineage expression (P < 0·01) as single cell CD34⁺CD45⁺GPA⁺ (P < 0·01) and CD34⁺CD45⁻GPA⁺ (P < 0·01) HSPCs. SCD BM is characterized by increased inflammation, aggregation and contamination contributing to significant differences in HSPC quality and yield compared to non-SCD BM.