Medical and ethical considerations on hematopoietic stem cells mobilization for healthy donors

2018 ◽  
Vol 25 (2) ◽  
pp. 136-143
Author(s):  
V. Moalic-Allain
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ethical Considerations ◽  
Hematopoietic Stem ◽  
Healthy Donors

scholarly journals Mobilization of Hematopoietic Stem Cells with Lenograstim in Healthy Donors: Efficacy and Safety Analysis According to Donor Age

2015 ◽  
Vol 21 (5) ◽  
pp. 881-888 ◽  
Author(s):  
Massimo Martino ◽  
Erminio Bonizzoni ◽  
Tiziana Moscato ◽  
Anna Grazia Recchia ◽  
Roberta Fedele ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Safety Analysis ◽  
Efficacy And Safety ◽  
Donor Age ◽  
Hematopoietic Stem ◽  
Healthy Donors

Malignant Myeloma Cells Impair Phenotype and Function of stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1799-1799
Author(s):  
Ingmar Bruns ◽  
Sebastian Büst ◽  
Akos G. Czibere ◽  
Ron-Patrick Cadeddu ◽  
Ines Brückmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Plasma Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
Stem And Progenitor Cells

Abstract Abstract 1799 Poster Board I-825 Multiple myeloma (MM) patients often present with anemia at the time of initial diagnosis. This has so far only attributed to a physically marrow suppression by the invading malignant plasma cells and the overexpression of Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by malignant plasma cells triggering the death of immature erythroblasts. Still the impact of MM on hematopoietic stem cells and their niches is scarcely established. In this study we analyzed highly purified CD34+ hematopoietic stem and progenitor cell subsets from the bone marrow of newly diagnosed MM patients in comparison to normal donors. Quantitative flowcytometric analyses revealed a significant reduction of the megakaryocyte-erythrocyte progenitor (MEP) proportion in MM patients, whereas the percentage of granulocyte-macrophage progenitors (GMP) was significantly increased. Proportions of hematopoietic stem cells (HSC) and myeloid progenitors (CMP) were not significantly altered. We then asked if this is also reflected by clonogenic assays and found a significantly decreased percentage of erythroid precursors (BFU-E and CFU-E). Using Affymetrix HU133 2.0 gene arrays, we compared the gene expression signatures of stem cells and progenitor subsets in MM patients and healthy donors. The most striking findings so far reflect reduced adhesive and migratory potential, impaired self-renewal capacity and disturbed B-cell development in HSC whereas the MEP expression profile reflects decreased in cell cycle activity and enhanced apoptosis. In line we found a decreased expression of the adhesion molecule CD44 and a reduced actin polymerization in MM HSC by immunofluorescence analysis. Accordingly, in vitro adhesion and transwell migration assays showed reduced adhesive and migratory capacities. The impaired self-renewal capacity of MM HSC was functionally corroborated by a significantly decreased long-term culture initiating cell (LTC-IC) frequency in long term culture assays. Cell cycle analyses revealed a significantly larger proportion of MM MEP in G0-phase of the cell cycle. Furthermore, the proportion of apoptotic cells in MM MEP determined by the content of cleaved caspase 3 was increased as compared to MEP from healthy donors. Taken together, our findings indicate an impact of MM on the molecular phenotype and functional properties of stem and progenitor cells. Anemia in MM seems at least partially to originate already at the stem and progenitor level. Disclosures Off Label Use: AML with multikinase inhibitor sorafenib, which is approved by EMEA + FDA for renal cell carcinoma.

scholarly journals Remobilization of hematopoietic stem cells in healthy donors for allogeneic transplantation

Transfusion ◽  
2016 ◽  
Vol 56 (9) ◽  
pp. 2331-2335 ◽  
Author(s):  
Mark A. Fiala ◽  
Soo Park ◽  
Michael Slade ◽  
John F. DiPersio ◽  
Keith E. Stockerl‐Goldstein
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Allogeneic Transplantation ◽  
Hematopoietic Stem ◽  
Healthy Donors

scholarly journals Whole Transcriptome Sequencing Identifies Novel Pathways Associated with Paroxysmal Nocturnal Hemoglobinuria- Increased CXCR2 Expression in PNH Granulocytes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3608-3608
Author(s):  
Kohei Hosokawa ◽  
Sachiko Kajigaya ◽  
Keyvan Keyvanfar ◽  
Danielle M. Townsley ◽  
Bogdan Dumitriu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Lymphoid Cells ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Cxcr2 Expression ◽  
Healthy Donors

Abstract Background. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder that arises from hematopoietic stem cells (HSCs). PNH is caused by a somatic mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A), responsible for a deficiency in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). PNH is a clonal disease that originates from HSCs, as the originating PIGA mutation is present in cells of multiple lineages, including myeloid, erythroid, and lymphoid cells. However, a critical question regarding PNH that has yet to be fully explained despite several decades of research is the mechanism responsible for clonal expansion of PIGA -mutant cells in bone marrow failure. Using RNA-seq, we identify pathways, coding and non-coding RNA transcripts, splice variants, or single nucleotide variants and other alterations that may relate to the selective advantage of PNH clone. Method. Blood samples were obtained after informed consent from patients with 14 PNH and 18 age-matched healthy donors. From PNH patients and healthy donors, 4 samples were used for RNA sequencing 6 samples were used for validation by flow cytometry. The liquid FLAER method was used for the detection of PNH-type granulocytes. For RNA extraction, granulocytes were sorted for CD11b+ FLAER+ granulocytes, CD11b+ FLAER- granulocytes. For bone marrow staining, cells not expressing lineage markers were separated into five subpopulations: Long-term hematopoietic stem cells (LT-HSC; Lin- CD34+ CD38- CD90+), short-term hematopoietic stem cells (ST-HSC; Lin- CD34+ CD38- CD90-), common myeloid progenitors (CMP; Lin- CD34+ CD38+ CD123+ CD45RA-), granulocyte-monocyte progenitors (GMP; Lin- CD34+ CD38+ CD123+ CD45RA+) and megakaryocyte-erythrocyte progenitors (MEP; Lin- CD34+ CD38+ CD123- CD45RA-). Results and Discussion. First, RNA expression levels in CD11b+ FLAER+ and CD11b+ FLAER- populations of PNH patients were analyzed using RNA sequencing. Expression levels of 7 mRNAs (CSF2RB, ACSL1, FCGR3B, IL1RN, CXCR2, TREM1, and TNFRSR10C) were significantly upregulated (> 3 FC, P < 0.01) in CD11b+ FLAER- cells of PNH patients compared with CD11b+ FLAER+ cells. To validate the differential expression observed in GPI-AP- granulocytes from PNH patients, protein expression levels of CSF2RB, FCGR3B, CXCR2, TREM1, and TNFRSF10C were assessed by flow cytometry. In CD11b+ FLAER- granulocytes of 6 PNH patients, increased expression of CXCR2 was validated, whereas decreased expression of FCGRB and TNFRSF10C were validated compared with CD11b+ FLAER+ granulocytes and that of healthy controls. Low expression FCGRB and TNFRSR10C in CD11b+ FLAER- granulocytes were considered to be reasonable, as these were GPI-APs. Next, we examined whether increased CXCR2 expression in PNH-type cells was validated in different peripheral blood cell populations. Increased CXCR2 expression in PNH-type cells was confirmed in granulocyte and monocyte populations, not in T cell or B cell population. We checked the expression levels of CXCR1 and CXCR2, which are closely related receptors that recognize CXC chemokines. CXCR2 expression was significantly different between normal and PNH-type cells in granulocytes and monocytes, and CXCR1 expression was significant only for granulocytes. To address the difference of CXCR2 expression levels between normal and PNH-type cells in more undifferentiated cells, we next examined the CXCR2 expression levels in bone marrow hematopoietic stem cells. Expression of CXCR2 was weakly expressed in hematopoietic stem cells and progenitors, both in normal and PNH-type cells, suggesting that difference of CXCR2 expression between normal and PNH-type cells is evident only in differentiated myeloid cells, not in hematopoietic stem cells or lymphoid cells. Conclusion. We provide evidence for increased expression of CXCR2 in PNH-type granulcoytes and monocytes by RNA-seq and flow cytometry. The differential expression of CXCR2 might partly explain the dominance of PNH clones in myeloid cells in patients. CXCR2 is an adverse prognostic factor in MDS/AML and is a potential therapeutic target against immature leukemic stem cell-enriched cell fractions in MDS and AML (Schinke C, et al, Blood, 2015). Understanding the mechanism of increased CXCR2 expression in PNH-type cells may offer new therapeutic strategies and novel mechanistic insight into the pathophysiology of PNH. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding. Dumitriu:Novartis: Research Funding; GSK: Research Funding. Young:Novartis: Research Funding; GSK: Research Funding.

scholarly journals 331: Effect of Smoking on Mobilization of Hematopoietic Stem Cells in Healthy Donors

2008 ◽  
Vol 14 (2) ◽  
pp. 122 ◽  
Author(s):  
A. Panwalkar ◽  
M. Devetten ◽  
F. Loberiza ◽  
M. Moehr ◽  
A. Kessinger
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Healthy Donors

Direct Confirmation of Quiescent Property of AML Stem Cells by Showing Markedly Decreased Plating Efficiency in Single Leukemic Stem Cell Culture

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4737-4737
Author(s):  
Myung-Geun Shin ◽  
Hye-Ran Kim ◽  
Chang-Soo Kim ◽  
Hyeoung-Joon Kim ◽  
Hoon Kook ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Plating Efficiency ◽  
Leukemic Stem Cell ◽  
Cell Sorter ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
Anti Cancer

Abstract Cancer stem cell, recently discovered to exist in colon cancer and brain tumor, is resistant to anti-cancer drugs and radiotherapy, demanding the development of new drugs and treatment strategies targeting tumor stem cells. Leukemic stem cell (LSC) has been accused to play a pivotal role in pathogenesis of hematological malignancy such as acute myeloblastic leukemia (AML). Various anti-cancer medicines, particularly anti-proliferative agents, have been ineffective in treating LSC due to its slower division process and longer interphase compared to normal stem cell and hematopoietic cell. This study comparatively examined growth and proliferation capacity (plating efficiency) of clonogenic hematopoietic progenitors and LSC from healthy donors and AML patients using single cell sorting and culture system (BD FACS Aria cell sorter; BD Biosciences, San Jose, CA). A total of 384 normal hematopoietic stem cells (CD34+CD38+/CD38−) were obtained from peripheral bloods and cord bloods donated by four donors using single cell sorter, and individual single cells were cultured in 96-well plates with each well containing 100ul of serum media, 100ng/ml of stem cell factor, 100ng/ml of Flt-3, 100ng/ml of thrombopoietin and 50ng/ml of G-CSF for five days. 768 single LSC (CD34+CD38−) and 384 single CD34+CD38+ cells were obtained from three AML patients. Growth and proliferation capacities of normal hematopoietic stem cell and LSC were determined in terms of plating efficiency (number of the wells in which more than two cells grew/total number of cells in 96-well plate culture × 100). Plating efficiency of individual normal single hematopoietic stem cells varied between samples. Eighty eight out of 192 single stem cells originated from cord blood grew into more than two cells, yielding plating efficiency of 45.8% and cells from the peripheral blood of two healthy donors 30.2% (58/192). In contrast, single LSC originated from the AML patients showed significantly lower plating efficiency with 14.6% (42/288), 3.6% (7/192) and 8.0% (23/288). These results directly confirmed quiescent and slowly dividing properties of LSC. In addition, plating efficiency of normal hematopoietic stem cells was shown to vary between their originating locations in healthy donors.

scholarly journals Alterations in Secretome and Transcriptome of Bone Marrow Derived MSCs in Patients with Diffuse Large B-Cell Lymphoma without Bone Marrow Involvement

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1514-1514
Author(s):  
Nataliya A. Petinati ◽  
Natalia Sats ◽  
Nina J. Drize ◽  
Irina Malyants ◽  
Victoria Shender ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Complete Remission ◽  
Hematopoietic Stem Cells ◽  
Signaling Pathways ◽  
B Cell Lymphoma ◽  
Ion Source ◽  
Phenol Red ◽  
Hematopoietic Stem ◽  
Healthy Donors

Introduction Multipotent mesenchymal stromal cells (MSCs) differentiate into all mesenchymal lineages, regulate hematopoietic stem cells, and also take part in immunomodulation. MSCs are damaged in patients with leukemia. Most of the patients with DLBCL do not have bone marrow (BM) involvement. Despite the absence of proved BM damage in DLBCL patients, the properties of MSCs are changed. We aimed to analyze secretome and transcriptome of MSCs derived from BM of DLBCL patients without BM involvement. Methods The study included 16 DLBCL patients (7 males and 9 females), of which 6 were 42-60-year-old in the onset of the disease and a month after the end of treatment with NHL-BFM90; 10 were 48-78-year-old in complete remission for 6-14 years (5 received CHOP and 5 NHL-BFM90 treatment). Control group included 5 healthy donors (3 males, 2 females), median age 37. During diagnostic punctures BM was collected after informed consent. MSCs were cultured by standard method. Confluent MSCs layers after 1 passage were cultivated in serum-free RPMI1640 without phenol red for 24 hours; supernatants were studied for secretome and cells for transcriptome. The analysis of MSCs secretome was carried out using the LC-MS/MS analysis (TripleTOF 5600+ mass spectrometer with a NanoSpray III ion source coupled to a NanoLC Ultra 2D+ nano-HPLC System. Total RNA was isolated, applying standard procedures, from MSCs. Next-generation sequencing of complementary DNA libraries of polyA-enriched RNA was performed with Illumina HiSeq. Raw RNA-seq data were processed using STAR. Gene expression was compared using the limma R/Bioconductor package. Results The total cell production for 4 passages in primary patients' MSCs was higher than in donors (26.6 ± 2 versus 10.1 ± 4.4 x 106 per flask). It remained elevated regardless of the time passed after therapy. The patterns of secretome and transcriptome of patients' MSCs differed dramatically from the MSCs of healthy donors (Table). In MSCs of primary patients, the secretion and transcription of proteins involved in IL-17, TNF and Toll-like receptor signaling pathways, cytokine-cytokine receptor interaction, cytokine-mediated signaling pathway, cellular response to cytokine stimulus, regulation of signaling receptor activity, regulation of neutrophil chemotaxis, inflammatory and acute inflammatory response and its regulation, leukocyte activation involved in immune response, immune system process, extracellular matrix organization were elevated. Secretion and transcription of cytokines and chemokines (IL6, IL4, LIF, TNFa, CXCL1 and CXCL3), taking part in hematopoiesis regulation were increased in primary patients MSCs. One month after treatment, secretion of 332 proteins was decreased, only 2 of them (DKK1 and FKBP7) were previously overexpressed in primary patients. Many years after the end of both variants of treatment, the secretion and transcription of 32 proteins participating in the same pathways as before treatment remains elevated compared with healthy donors. In addition, the complement and coagulation cascades became upregulated. In MSCs of all patients, regardless of therapy and remission duration , expression/ secretion of following genes/proteins: ACAN, COL1A, MMP3, TGFb1, NDNF, CANX, LAP3, MGP, SERPINB2, STC1,TFPI,TMEM132A, BMP2, CFH, HILPDA, IDO1, IL1B, ITGA2, JUN, LMO2, MMP13, MMP3, TNFRSF1B,TNFSF4 was increased. Some of these proteins take part in bone and cartilage formation, hematopoietic stem cells regulation, blood coagulation and inflammation. These changes in secreted proteins reflect the response of MSCs at the organism level to the tumor presence. Moreover, NUCKS1 overexpression was observed in MSCs of all patients. This nuclear casein kinase plays a significant role in modulating chromatin structure and regulates replication, transcription, and chromatin condensation. Furthermore, this protein contributes to the susceptibility, occurrence, and development of several types of cancer and other diseases. NUCKS1 is considered to be a potent marker for such diseases. Conclusion The presence of a lymphoid tumor without BM involvement in the body leads to irreversible changes in the BM MSCs, thus affecting a lot of biological processes and signaling pathways, independent of the treatment and duration of complete remission. The work were supported by the Russian Foundation for Basic Research, Project No. 17-00-00170. Disclosures No relevant conflicts of interest to declare.

PIG-A Mutations in Normal Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2825-2825
Author(s):  
Rong Hu ◽  
Galina Mukhina ◽  
Steven Piantadosi ◽  
Richard J. Jones ◽  
Robert A. Brodsky
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Genetic Fidelity ◽  
Large Mammals ◽  
Hematopoietic Stem ◽  
Differentiated Cells ◽  
Mutational Frequency ◽  
Healthy Donors ◽  
Normal Individuals

Abstract Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is caused by somatic mutations of the X-linked gene, PIG-A, in hematopoietic stem cells (HSCs). The product of this gene is necessary for the assembly of glycosylphosphatidylinositol (GPI) anchors. Consequently, PNH cells lack the expression of GPI-anchored proteins on their cell surface. PIG-A mutations have been found in granulocytes and T lymphocytes from most normal individuals. Although the significance of these mutations is unclear, it suggests that they are important in the pathogenesis of PNH. Methods: We isolated CD34+ progenitors from 4 PNH patients, 18 healthy donors, and 9 non-PNH patients undergoing peripheral blood stem cell tranplantation. The frequency of PIG-A mutant progenitors was determined by assaying for colony forming cells (CFC) in methylcellulose containing toxic doses of aerolysin. Aerolysin is a pore-forming toxin that uses the GPI anchor as it receptor; hence, PNH cells are unique in their resistance to aerolysin. DNA was extracted from individual day 14 aerolysin resistant CFC and the PIG-A gene was sequenced to determine clonality. We performed a Poisson distribution of the mutational frequency to determine the probability that the PIG-A mutation in controls arose from HSC. Results: In PNH patients, 67% of the CFC were aerolysin resistant. The frequency of aerolysin resistant CFC was 14.7 ± 4.0 x 10−6 in the bone marrow of healthy donors and was 57.0 ± 6.7 x 10−6 from mobilized peripheral blood. Aerolysin resistant CFC from PNH patients exhibited clonalPIG-A mutations, and thus, arose from HSC. In contrast, PIG-A mutations in the CFC from controls were polyclonal (up to 15 different mutations from one individual). Recent evidence suggests that humans and other large mammals possess only 10,000 primitive HSC, and that only 1000 of these cells are thought to contribute to hematopoiesis at any one time. Poisson statistics show that only 5% of normals would be expected to harbor a PIG-A mutation in 1000 HSC, and &lt; 1 x 10−9 persons would harbor 10 or more different PIG-A mutations even if all 10,000 hematopoietic stem cells were contributing to hematopoiesis. Thus, the high frequency of PIG-A mutations in controls, coupled with their polyclonality, suggests that they do not arise at the level of HSC; rather, PIG-A mutations in normals appear to arise as a consequence of hematopoietic differentiation, between the level of an HSC and a CFC. Conclusion: Our data confirm the findings that PIG-A mutations are relatively common in normal hematopoiesis. Although we cannot rule out that a rare PIG-A mutant blood cell in normals does in fact arise from a mutant HSC, our data suggest that most of the mutations occur with differentiation. Genetic fidelity can be lost with differentiation without consequence, as mutations in differentiated cells would not be propagated. These data also call into question the relevance of PIG-A mutations in normals to the pathogenesis of PNH.

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