Circulating hematopoietic progenitor cells in first trimester fetal blood

Blood ◽  
2000 ◽  
Vol 95 (6) ◽  
pp. 1967-1972 ◽  
Author(s):  
Cesare Campagnoli ◽  
Nicholas Fisk ◽  
Timothy Overton ◽  
Phillip Bennett ◽  
Timothy Watts ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
First Trimester ◽  
Fetal Life ◽  
Fetal Blood ◽  
Maternal Circulation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The yolk sac and aorto-gonad-mesonephros region are well recognized as the principal sites of hematopoiesis in the developing embryo, and the liver is the principal site of hematopoiesis in the fetus. However, little is known about circulating hematopoietic stem and progenitor cells in early fetal life. We investigated the number and characteristics of circulating progenitors in first trimester blood of 64 human fetuses (median gestational age, 10+4 weeks; range, 7+6-13+6 weeks). CD34+ cells accounted for 5.1 ± 1.0% of CD45+ cells in first trimester blood, which is significantly more than in term cord blood (0.4 ± 0.03%;P = .0015). However, the concentration of CD34+ cells (6.6 ± 2.4 × 104/mL) was similar to that in term cord blood (5.6 ± 3.9 × 104/mL). The total number of progenitors cultured from unsorted mononuclear cells (MNCs) in first trimester blood was 19.2 ± 2.1 × 103/mL, which is similar to that in term cord blood (26.4 ± 5.6 × 103/mL). All lineages were seen: colony-forming unit–GEMM (CFU-GEMM), CFU-GM, BFU-e, BFU-MK, and CFU-MK. Clonogenic assays of CD34+ cells purified from first trimester samples produced mainly two lineages: BFU-e (39.0 ± 9.6 × 103/mL CD34+ cells) and CFU-GEMM (22.6 ± 4.7 × 103/mL CD34+ cells). Short-term liquid culture of first trimester blood MNCs in SCF + IL-3 + Flt-3 (stem cell factor + interleukin-3 + Flt-3) increased, by 7-fold, the numbers of CFU-GEMM and induced a dramatic increase in BFU-e (65.6 ± 12.1–fold). These data show that significant numbers of committed and multipotent progenitors with capacity for expansion circulate in first trimester fetal blood and can be CD34 selected. These cells should be suitable targets for gene transfer and stem cell transplantation and, because fetal hematopoietic progenitors have been demonstrated in the maternal circulation from early gestation, may also be manipulated for noninvasive prenatal diagnosis of major genetic disorders.

scholarly journals Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34+Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Hui Xie ◽  
Li Sun ◽  
Liming Zhang ◽  
Teng Liu ◽  
Li Chen ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Promising Therapeutic Approach ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

scholarly journals High Integrity and Fidelity of Long-Term Cryopreserved Umbilical Cord Blood for Transplantation

2021 ◽  
Vol 10 (2) ◽  
pp. 293
Author(s):  
Gee-Hye Kim ◽  
Jihye Kwak ◽  
Sung Hee Kim ◽  
Hee Jung Kim ◽  
Hye Kyung Hong ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Clinical Applications ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Hsc Transplantation

Umbilical cord blood (UCB) is used as a source of donor cells for hematopoietic stem cell (HSC) transplantation. The success of transplantation is dependent on the quality of cord blood (CB) units for maximizing the chance of engraftment. Improved outcomes following transplantation are associated with certain factors of cryopreserved CB units: total volume and total nucleated cell (TNC) count, mononuclear cell (MNC) count, and CD34+ cell count. The role of the storage period of CB units in determining the viability and counts of cells is less clear and is related to the quality of cryopreserved CB units. Herein, we demonstrate the recovery of viable TNCs and CD34+ cells, as well as the MNC viability in 20-year-old cryopreserved CB units in a CB bank (MEDIPOST Co., Ltd., Seongnam-si, Gyeonggi-do, Korea). In addition, cell populations in CB units were evaluated for future clinical applications. The stable recovery rate of the viability of cryopreserved CB that had been stored for up to 20 years suggested the possibility of uses of the long-term cryopreservation of CB units. Similar relationships were observed in the recovery of TNCs and CD34+ cells in units of cryopreserved and fresh CB. The high-viability recovery of long-term cryopreserved CB suggests that successful hematopoietic stem cell (HSC) transplantation and other clinical applications, which are suitable for treating incurable diseases, may be performed regardless of long-term storage.

scholarly journals Very Small Embryonic-Like Stem Cells, Endothelial Progenitor Cells, and Different Monocyte Subsets Are Effectively Mobilized in Acute Lymphoblastic Leukemia Patients after G-CSF Treatment

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Andrzej Eljaszewicz ◽  
Lukasz Bolkun ◽  
Kamil Grubczak ◽  
Malgorzata Rusak ◽  
Tomasz Wasiluk ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Lymphoblastic Leukemia ◽  
Monocyte Subsets ◽  
Endothelial Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Background. Acute lymphoblastic leukemia (ALL) is a malignant disease of lymphoid progenitor cells. ALL chemotherapy is associated with numerous side effects including neutropenia that is routinely prevented by the administration of growth factors such as granulocyte colony-stimulating factor (G-CSF). To date, the effects of G-CSF treatment on the level of mobilization of different stem and progenitor cells in ALL patients subjected to clinically effective chemotherapy have not been fully elucidated. Therefore, in this study we aimed to assess the effect of administration of G-CSF to ALL patients on mobilization of other than hematopoietic stem cell (HSCs) subsets, namely, very small embryonic-like stem cells (VSELs), endothelial progenitor cells (EPCs), and different monocyte subsets. Methods. We used multicolor flow cytometry to quantitate numbers of CD34+ cells, hematopoietic stem cells (HSCs), VSELs, EPCs, and different monocyte subsets in the peripheral blood of ALL patients and normal age-matched blood donors. Results. We showed that ALL patients following chemotherapy, when compared to healthy donors, presented with significantly lower numbers of CD34+ cells, HSCs, VSELs, and CD14+ monocytes, but not EPCs. Moreover, we found that G-CSF administration induced effective mobilization of all the abovementioned progenitor and stem cell subsets with high regenerative and proangiogenic potential. Conclusion. These findings contribute to better understanding the beneficial clinical effect of G-CSF administration in ALL patients following successful chemotherapy.

Impaired Alloantigen Presenting Activity of Cord Blood Nucleated Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2203-2203
Author(s):  
Sandeep Chunduri ◽  
Dolores Mahmud ◽  
Javaneh Abbasian ◽  
Damiano Rondelli
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Solid Organ Transplantation ◽  
T Cell Response ◽  
Solid Organ ◽  
Cd34 Cells

Abstract Transplantation of HLA-mismatched cord blood (CB) nucleated cells has limited risk of severe acute graft-versus-host disease and graft rejection. This may depend on naïve T cells not yet exposed to many antigens and on immature antigen-presenting cells (APC) not delivering appropriate signals to allogeneic T cells. In order to test the APC activity of human circulating CB cells in-vitro, we initially used irradiated CB mononuclear cells (MNC) or immunomagnetically selected CD34+ cells, CD133+ cells, or CD14+ monocytes to stimulate the proliferative response of incompatible blood T cells in mixed leukocyte culture (MLC). CB MNC failed to induce allogeneic T cell proliferation, while CD34+ and CD133+ progenitors or CD14+ monocytes induced potent T cell alloresponses. Nevertheless, since allogeneic T cell response was not restored after depletion of CD3+ cells in the CB, nor the add-back of irradiated CB MNC to CD34+ or CD14+ stimulators inhibited allo-T cells, a direct suppressive effect of CB MNC was excluded. Allogeneic peripheral blood cytotoxic T-lymphocyte (CTL) responses were not induced after 7 days of stimulation with irradiated CB MNC, although after 4 weekly rechallenges with CB MNC, on average a 23% lysis of antigen-specific CB PHA-blasts was observed at the highest effector:target ratio (50:1). To test the tolerogenic potential of CB MNC, T cells initially exposed to CB MNC were rechallenged in secondary MLC with CB MNC, or CD34+ cells, or monocyte-derived dendritic cells (Mo-DC) generated in liquid culture with GM-CSF and IL-4. Allogeneic T cells were still unresponsive upon rechallenge with CB MNC, but proliferated upon 3 days of restimulation with CD34+ cells or Mo-DC from the same CB. Surprisingly, the supernatant of these latter MLCs did inhibit completely a 3rd party MLC. Instead, the supernatant of blood T cells that had been activated by CB CD34+ cells or Mo-DC both in primary and secondary MLC did not. These results show an impaired allo-APC activity of CB MNC but not CB CD34+ cells, and suggest that T cells releasing immunosuppressive cytokines may be activated by CB MNC and then expanded by a second more potent stimulation with professional APC. This hypothesis could explain the sustained engraftment of HLA-mismatched CB stem cell transplants in humans. Based on these results, the in-vivo or ex-vivo downregulation of T cell alloreactivity induced by CB MNC will be tested in experimental models of stem cell, as well as solid organ transplantation.

Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4040-4040
Author(s):  
Szabolcs Fatrai ◽  
Simon M.G.J. Daenen ◽  
Edo Vellenga ◽  
Jan J. Schuringa
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Fold Increase ◽  
Marrow Stromal Cells ◽  
Muc1 Expression ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.

Characterization of the Putative Stem Cells From Umbilical Cord Blood.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4231-4231
Author(s):  
Amos S. Gaikwad ◽  
Michael Cubbage ◽  
Tatiana Goltsova ◽  
Christopher Threeton ◽  
Maria Ty ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Cell Population ◽  
Cell Biology ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Side Population ◽  
Hematopoietic Stem ◽  
Cell Fractions

Abstract Abstract 4231 Cord blood (CB) is a rich source of hematopoietic stem cells (HSC) with long-term repopulating activity necessary for allogeneic stem cell transplantation. CD34+ stem cells are considered sufficient for transplantation, however recent progress in stem cell biology indicates that cells with other surface markers such as CD133 or cells expressing high aldehyde dehydrogenase activity with low side scatter (ALDHhigh/SSClow) or a rare side population (SP) of cells that exclude the Hoechst 33342 vital dye via multi drug transporters have been shown to possess stem cell properties. We characterized CD34+, CD133+, ALDH+ and SP in mononuclear cells (MNC) isolated from human CB. While the SP cell population is rare and detectable in few CB-MNC examined, we found abundant CD34+ and CD133+ cells (1.0+/-0.5 and 0.8+/-0.4 per 100 CD45+ MNC cells, respectively) following the ISHAGE protocol. A distinct ALDH+ cell population (median of 0.26%; range of 0.1 to 0.5%) was also present in all of the CB-MNC analyzed. Over 90% of the ALDH+ cells were also CD34+ and CD133+. The ability of CB-MNC to form colonies in methocult semi-solid media supplemented with cytokines yielded myeloid, lymphoid and erythroid colonies. The clonogenic potential of CB-MNC ranged from 16-48%. We are assessing the colony forming ability of purified stem cell fractions using flow cytometry. The clonogenic efficiency of these individual putative stem cells will be discussed. Disclosures: No relevant conflicts of interest to declare.

Long-Term Reconstitution of Transduced Rhesus CD34+ Cells Mobilized by G-CSF and Plerixafor.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1449-1449
Author(s):  
Naoya Uchida ◽  
Aylin Bonifacino ◽  
Allen E Krouse ◽  
Sandra D Price ◽  
Ross M Fasano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Transgene Expression ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Cd34 Cells ◽  
One Year

Abstract Abstract 1449 Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor (AMD3100) produces significant mobilization of peripheral blood stem cells in the rhesus macaque model. The CD34+ cell population mobilized possesses a unique gene expression profile, suggesting a different proportion of progenitor/stem cells. To evaluate whether these CD34+ cells can stably reconstitute blood cells, we performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34+ cells that were transduced with chimeric HIV1-based lentiviral vector including the SIV-capsid (χHIV vector). In our experiments, G-CSF and plerixafor mobilization (N=3) yielded a 2-fold higher CD34+ cell number, compared to that observed for G-CSF and stem cell factor (SCF) combination (N=5) (8.6 ± 1.8 × 107 vs. 3.6 ± 0.5 × 107, p<0.01). Transduction rates with χHIV vector, however, were 4-fold lower in G-CSF and plerixafor-mobilized CD34+ cells, compared to G-CSF and SCF (13 ± 4% vs. 57 ± 5%, p<0.01). CD123+ (IL3 receptor) rates were higher in CD34+ cells mobilized by G-CSF and plerixafor (16.4%) or plerixafor alone (21.3%), when compared to G-CSF alone (2.6%). To determine their repopulating ability, G-CSF and plerixafor-mobilized CD34+ cells were transduced with EGFP-expressing χHIV vector at MOI 50 and transplanted into lethally-irradiated rhesus macaques (N=3). Blood counts and transgene expression levels were followed for more than one year. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages and earlier recovery of lymphocytes, compared to animals who received G-CSF and SCF-mobilized grafts (1200 ± 300/μl vs. 3300 ± 900/μl on day 30, p<0.05). One month after transplantation, there was a transient development of a skin rash, cold agglutinin reaction, and IgG and IgM type plasma paraproteins in one of the three animals transplanted with G-CSF and plerixafor cells. This animal had the most rapid lymphocyte recovery. These data suggested that G-CSF and plerixafor-mobilized CD34+ cells contained an increased amount of early lymphoid progenitor cells, compared to those arising from the G-CSF and SCF mobilization. One year after transplantation, transgene expression levels were 2–5% in the first animal, 2–5% in the second animal, and 5–10% in the third animal in all lineage cells. These data indicated G-CSF and plerixafor-mobilized CD34+ cells could stably reconstitute peripheral blood in the rhesus macaque. Next, we evaluated the correlation of transgene expression levels between in vitro bulk CD34+ cells and lymphocytes at one month, three months, and six months post-transplantation. At one and three months after transplantation, data from G-CSF and plerixafor mobilization showed higher ratio of %EGFP in lymphocytes to that of in vitro CD34+ cells when compared to that of G-CSF and SCF mobilization. At six months after transplantation the ratios were similar. These results again suggest that G-CSF and plerixafor-mobilized CD34+ cells might include a larger proportion of early lymphoid progenitor cells when compared to G-CSF and SCF mobilization. In summary, G-CSF and plerixafor mobilization increased CD34+ cell numbers. G-CSF and plerixafor-mobilized CD34+ cells contained an increased number of lymphoid progenitor cells and a hematopoietic stem cell population that was capable of reconstituting blood cells as demonstrated by earlier lymphoid recovery and stable multilineage transgene expression in vivo, respectively. Our findings should impact the development of new clinical mobilization protocols. Disclosures: No relevant conflicts of interest to declare.

CD34+ Circulating Progenitor Cell Frequency Is Highly Heritable

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4309-4309
Author(s):  
Kenneth S. Cohen ◽  
Susan Cheng ◽  
Martin G. Larson ◽  
L. Adrienne Cupples ◽  
Elizabeth L. McCabe ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Stem Cell ◽  
Cigarette Smoking ◽  
Framingham Risk Score ◽  
Mononuclear Cells ◽  
Cell Frequency ◽  
Hematopoietic Stem ◽  
Framingham Risk ◽  
Cd34 Cells ◽  
Clinical Covariates

Abstract Abstract 4309 Background: Circulating blood CD34+ cells are a heterogeneous population of cells comprising hematopoietic stem/progenitor cells, circulating angiogenic cells, and a small population of endothelial/endothelial progenitor cells. CD34+ cells are used clinically for hematopoietic stem cell transplants and have also been studied as cellular biomarkers of response to anti-angiogenic therapy in clinical trials and as markers of cardiovascular disease (CVD) risk. Understanding factors that regulate circulating CD34+ cell frequency has important implications for the fields of stem cell transplantation, oncology, cardiology, and vascular biology. Therefore, we initiated a large, community-based cohort study to examine clinical characteristics associated with circulating CD34+ cell frequency, as well as to define the heritable contribution to this phenotype. Methods: We assessed CD34+ cell frequency in 1,786 participants attending the eighth examination cycle (2005-2008) of the Framingham Offspring Study (mean age 66 years, 54% women). 1,595 participants had no known history of cardiovascular disease. Participants underwent a standardized medical examination and laboratory assessment of cardiovascular risk factors. These included systolic and diastolic blood pressure, body mass index, glucose, total and high-density lipoprotein, cholesterol, triglycerides, medications (lipid-lowering, antihypertensive, and hormone replacement therapies), cigarette smoking, and diabetes (defined as a fasting glucose >126 mg/dL or the use of medications to treat diabetes). For CD34+ cell phenotyping, buffy coat samples were diluted with PBS and mononuclear cells isolated by Ficoll density-gradient centrifugation. Mononuclear cells were stained with an anti-human CD34 antibody and cells were analyzed by FACS analysis (Becton-Dickenson FACSCalibur). Data was analyzed as the frequency of CD34+ events within the cellular gate. Results: In multivariable-adjusted analyses adjusting for familial (sibling) correlations, CD34+ frequency was inversely associated with older age, female gender, and cigarette smoking. CD34+ frequency was positively associated with weight, total cholesterol, and statin therapy. Framingham risk score was inversely correlated with CD34+ frequency in men (coefficient -0.037, P=0.004) but not in women (coefficient 0.08, P=0.59). CD34+ frequency was not associated with prevalent CVD (P=0.69). Notably, the clinical covariates associated with CD34+ frequency only accounted for 6.3% of CD34+ variability. Therefore, we performed an assessment of CD34+ frequency as a heritable trait. Using variance component methods implemented in SOLAR on sibship-pair and spouse-pair data, the estimated heritability (h2) for CD34+ cell variability was 53.9 +/− 9.2 % (mean +/− standard error) in age- and sex-adjusted analyses and was highly statistically significant (P<0.00001). Conclusions: This study represents the largest assessment of clinical correlates of circulating CD34+ cell frequency to date. The inverse correlation between CD34+ cell frequency and Framingham risk score in a population predominantly free of CVD was confirmed; however, this finding was limited to male study subjects, and was driven by the inverse association with age. Surprisingly, previously identified clinical covariates accounted for only 6.3% of CD34+ variability, while the proportion of unexplained variance attributable to heritable factors accounted for 53.9% of CD34+ cell variability. These data, therefore, support efforts to define genetic variants that may regulate CD34+ frequency. Such investigations would have important implications for the fields of stem cell transplantation, as well as the study of hematologic and cardiovascular diseases more broadly. Disclosures: Martin: Genzyme: Employment.

The Tetraspanin CD9 Regulates Engraftment and Mobilization of Human CD34+ Hematopoietic Stem/Progenitor Cells and Modulates VLA-4 Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1169-1169
Author(s):  
Kam Tong Leung ◽  
Karen Li ◽  
Yorky Tsin Sik Wong ◽  
Kathy Yuen Yee Chan ◽  
Xiao-Bing Zhang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Scid Mouse ◽  
Conflicts Of Interest ◽  
Chimeric Mice ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells

Abstract Migration, homing and engraftment of hematopoietic stem/progenitor cells depend critically on the SDF-1/CXCR4 axis. We previously identified the tetraspanin CD9 as a downstream signal of this axis, and it regulates short-term homing of cord blood (CB) CD34+ cells (Leung et al, Blood, 2011). However, its roles in stem cell engraftment, mobilization and the underlying mechanisms have not been described. Here, we provided evidence that CD9 blockade profoundly reduced long-term bone marrow (BM; 70.9% inhibition; P = .0089) and splenic engraftment (87.8% inhibition; P = .0179) of CB CD34+ cells (n = 6) in the NOD/SCID mouse xenotransplantation model, without biasing specific lineage commitment. Interestingly, significant increase in the CD34+CD9+ subsets were observed in the BM (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4), indicating that CD9 expression on CD34+ cells is up-regulated during engraftment in the SDF-1-rich hematopoietic niches. Analysis of paired BM and peripheral blood (PB) samples from healthy donors revealed higher CD9 expressions in BM-resident CD34+ cells (46.0% CD9+ cells in BM vs 26.5% in PB; n = 13, P = .0035). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (32.3% CD9+ cells; n = 25), when compared with those in BM (47.7% CD9+ cells; n = 16, P = .0030). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.5-fold increase; n = 4, P = .0060). Treatment of NOD/SCID chimeric mice with G-CSF decreased the CD34+CD9+ subsets in the BM from 79.2% to 62.4% (n = 8, P = .0179). These data indicate that CD9 expression is down-regulated during egress or mobilization of CD34+ cells. To investigate the possible mechanisms, we performed a VCAM-1 (counter receptor of the VLA-4 integrin) binding assay on BM CD34+ cells. Our results demonstrated that CD34+CD9+ cells preferentially bound to soluble VCAM-1 (17.2%-51.4% VCAM-1-bound cells in CD9+ cells vs 12.8%-25.9% in CD9- cells; n = 10, P ≤ .0003), suggesting that CD9+ cells possess higher VLA-4 activity. Concomitant with decreased CD9 expression, MPB CD34+ cells exhibited lower VCAM-1 binding ability (2.8%-4.0% VCAM-1-bound cells; n = 3), when compared to BM CD34+ cells (15.5%-37.7%; n = 10, P < .0130). In vivo treatment of NOD/SCID chimeric mice with G-CSF reduced VCAM-1 binding of CD34+ cells in the BM by 49.0% (n = 5, P = .0010). Importantly, overexpression of CD9 in CB CD34+ cells promoted VCAM-1 binding by 39.5% (n = 3, P = .0391), thus providing evidence that CD9 regulates VLA-4 activity. Preliminary results also indicated that enforcing CD9 expression in CB CD34+ cells could enhance their homing and engraftment in the NOD/SCID mouse model. Our findings collectively established that CD9 expression and associated integrin VLA-4 activity are dynamically regulated in the BM microenvironment, which may represent important events in governing stem cell engraftment and mobilization. Strategies to modify CD9 expression could be developed to enhance engraftment or mobilization of CD34+ cells. Disclosures No relevant conflicts of interest to declare.

Ciclopirox Ethanolamine Is a Novel Modifier of Human Hematopoietic Stem Cell Ex Vivo Expansion

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 36-36
Author(s):  
Mehrnaz Safaee Talkhoncheh ◽  
Fredrik Ek ◽  
Aurelie Baudet ◽  
Christine Karlsson ◽  
Roger Olsson ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Cell Activity ◽  
Cell Number ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem Cell Activity ◽  
Cells Cultured

Abstract Despite extensive studies over the last decades, little is known about the mechanisms governing human hematopoietic stem cell (HSC) fate decisions. In particular, it has been challenging to define culture conditions in which HSCs can be expanded for clinical benefit. Application of small molecule screening to modulate stem cells has emerged as a useful tool for identification of new compounds with ability to expand hematopoietic stem and progenitor cells (HSPCs). Such screens have mainly relied on the expression of CD34 as predictor of stem cell activity in cultured cells. However, CD34 defines a broad repertoire of progenitor cells and does not define stem cell function. We found that the long-term repopulation potential of cultured human HSPCs is exclusively contained within a discrete cell population co-expressing CD34 and CD90, while the vast majority of progenitor cells are found in the CD34+CD90- population. Tracking the CD34+ CD90+ population is therefore a sensitive and specific tool to predict stem cell activity in cultured hematopoietic cells and provides a good basis for a screen aimed at discovering modifiers of stem cell expansion. To search broadly for novel and potential modifiers of ex vivo HSCs expansion we next developed and optimized a small molecule screen in human cord blood (CB) derived CD34+ cells. We screened >500 small molecules from 8 different annotated chemical libraries for the phenotypic expansion of CD34+ CD90+ cells following a 6-day culture in serum-free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and fms-like tyrosine kinase 3 ligand (FL). The numbers of CD34+ CD90+ cells for each molecule, tested at two different concentrations, was compared to DMSO treated controls. Following the initial screen, several candidate hits were selected and subjected to a dose response validation experiment from which we selected four top candidate molecules. Two of these molecules were histone deacetylase (HDAC) inhibitors, which recently have been reported to facilitate expansion of CB derived HSCs. One of the top candidates, Ciclopirox ethanolamine (CE), had previously not been implicated in HSC expansion. Ciclopirox ethanolamine is known as an antifungal agent and iron chelator. It has further been shown to suppress cancer cell survival through inhibition of Wnt/beta catenin signaling. We found that CB cells cultured with CE had a 4-fold increase in CD34+90+ cell number compared to DMSO treated controls following 6 days of culture. Interestingly, the total cell count was not different, suggesting a specific increase in CD34+ CD90+ cell number rather than an overall higher proliferation rate. When plated in methylcellulose, CE cultured cells generated increased numbers of myeloid colonies. Moreover, CE treated cells gave rise to multilineage colonies (CFU-GEMM) that could not be detected from the control cultures. To further test the functional capacity of cells cultured with CE, we transplanted cultured equivalents of 30,000 CB CD34+ cells (cultured with or without CE) into sub lethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human hematopoietic reconstitution in peripheral blood was determined 16 weeks later. Mice transplanted with CE cultured cells showed higher human CD45 engraftment 16 weeks post transplant compared to control cells (33.2±6.7% vs 14.6±5% p=0.04). The engrafted cells contributed to both myeloid and lymphoid lineages. This shows that Ciclopirox ethanolamine enhances the long-term engraftment capacity of ex vivo cultured HSCs and suggests that it should be considered in stem cell expansion protocols, either alone or in combination with other molecules. We are currently addressing the basis for the increased stem cell activity mediated by Ciclopirox ethanolamine using parameters for differentiation, cell cycling and apoptosis. In addition, we are comparing Ciclopirox ethanolamine with other recently defined modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document