scholarly journals Intravitreal Use of a Bone Marrow Mononuclear Fraction (BMMF) Containing CD34+ Cells in Patients with Stargardt Type Macular Dystrophy

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Carina Costa Cotrim ◽  
André M. Vieira Messias ◽  
Rodrigo Jorge ◽  
Rubens Camargo Siqueira
Keyword(s):  
Bone Marrow ◽  
Visual Acuity ◽  
Therapeutic Potential ◽  
Treated Group ◽  
Untreated Group ◽  
Macular Dystrophy ◽  
Full Field ◽  
Sham Injection ◽  
Cd34 Cells ◽  
Significant Difference

To assess the therapeutic potential and the safety of intravitreous use of a bone marrow mononuclear fraction (BMMF) containing CD34+ cells in patients with Stargardt type macular dystrophy. The study was conducted on 10 patients with Stargardt dystrophy with worse eye   visual   acuity ≤ 20 / 125 . A bone marrow aspirate was obtained from all patients, and after processing in the cell therapy center (CTC), 0.1 ml of the intravitreous BMMF suspension was injected into the eye with worse visual acuity. A sham injection was performed in the contralateral eye. The patients were evaluated at baseline and one, three, and six months after the injection. All of them were submitted to measurement of best corrected visual acuity (BCVA), microperimetry, multifocal electroretinography (mfERG) and full field electroretinography (ffERG), autofluorescence (AF), and optical coherence tomography (OCT). Fluorescein angiography was also performed before and six months after the injection. All patients completed the six-month period of evaluation. Mean visual acuity of the treated eye was 1.1 logMAR (20/250) before intravitreous (IV) injection, 0.96 logMAR (20/200+2) one month after injection, and 0.92 logMAR (20/160-1) 3 months after injection. In the untreated eye, mean VA was 1.0 logMAR (20/200) at baseline and 0.96 logMAR (20/200+2) and 0.94 logMAR (20/160-2) one and three months after injection, respectively. In the treated group, VA at baseline ranged from best acuity of 20/125-1 to worst acuity of 20/640+2, going through 20/100+2 and 20/400 during the first month. In the untreated group, BCVA ranged from 20/100+2 to 20/400 at baseline and from 20/100 to 20/400 after one month. The results for the treated group differed significantly at all follow-up times, whereas no significant difference was observed in the untreated group. Regarding the mean sensitivity of microperimetry, although there was improvement throughout all months, a significant difference occurred only during the first month. In the untreated eye, there was no significant difference in any analysis. Angiofluoresceinography did not reveal neovessel formation or tumor growth. The remaining exams were used in order to aid the diagnosis. The results indicate that the use of intravitreous BMMF in patients with Stargardt dystrophy is safe and is associated with a discrete improvement of BCVA and microperimetry in the treated eye compared to the untreated one.

Genotypic Representation of Myelodysplastic/Myeloproliferative Neoplasms in Nrg, Nrg-3GS and Srg-W41 Mice with Transgenic Expression of Human Cytokines

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2038-2038
Author(s):  
Hein Than ◽  
Naoto Nakamichi ◽  
Anthony D. Pomicter ◽  
John O'Shea ◽  
Orlando Antelope ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Myeloproliferative Neoplasms ◽  
Model Systems ◽  
Juvenile Myelomonocytic Leukemia ◽  
Functional Screening ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Myelomonocytic Leukemia

Abstract Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are complex clonal hematopoietic stem cell malignancies with overlapping dysplastic and proliferative features. Genomic analyses have charted the somatic mutation spectrum of MDS/MPN and revealed a major role for epigenetic dysregulation in their pathogenesis. No disease-modifying therapies are currently available, as progress has been hampered by a lack of genetically faithful in vivo model systems suitable for the preclinical development of new strategies. Yoshimi et al (Blood. 2017;130:397-407) recently showed that patients' chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) cells transplanted into NOD/SCID-IL2Rγ-/-mice expressing human IL3, GM-CSF and SCF transgenes (NSG-3GS mice) produced xenografts that had mutations characteristic of the input cells. Since we had demonstrated a superior level of chimerism achieved from transplants of normal human CD34+cord blood cells in SirpaNOD/Rag1-/-/IL2rγc-/-/W41/41mice with c-KIT deficiency (with an otherwise mixed NOD-C57Bl/6 background - SRG-W41 mice) compared to conventional NSG or NRG hosts (Miller et al. Exp Hematol. 2017;48:41-49), it was of interest to explore their use as hosts of samples from patients with MDS/MPN: CMML, atypical chronic myeloid leukemia (aCML) and secondary acute myeloid leukemia (sAML) progressed from CMML or aCML. Heparinized blood or bone marrow samples were obtained from patients treated at Huntsman Cancer Institute after informed consent. Diagnoses included CMML (n=5), aCML (n=2), and sAML (n=2). Unseparated cells were shipped by overnight courier to Vancouver and CD34+cells isolated on the same day were injected intravenously into sub-lethally irradiated female NRG mice or SRG-W41 mice, or in some cases the same sex and strains also carrying the human 3GS transgenes (NRG-3GS or SRG-W41-3GS mice) in accordance with British Columbia Cancer Agency institutional guidelines. Occasionally when mice were not immediately available, or large numbers of cells were available, cells were viably cryopreserved and transplanted later after thawing. Mice were observed for up to 36 weeks after xenotransplantation with .05 to 1.1x106 human CD34+cells. Engraftment of human CD45+cells in xenografts was evaluated by immunophenotyping, and a median of 90% human chimerism (range: 1% - 95%) was achieved at the time of bone marrow harvest from xenografts. Variant allele frequencies (VAF) were determined in genomic DNA extracted from both the patient samples (CD34+cells) and matching fluorescence-activated cells (FACS)-sorted human CD45+cells (hCD45+cells) purified from xenografts (1-5 xenografts per patient sample). DNA samples were subjected to PCR amplification with extension primers and analyzed using a MALDI-TOF mass spectrometer (MassArray, Agena Bioscience, San Diego, CA). Each mutation call was assigned by the software based on the molecular weight of the extended primer. Analysis of hCD45+cells from eight xenograft samples so far demonstrated a strong correlation of VAF between the patient samples and hCD45+cells from xenografts, in both SRG-W41-3GS (R2=0.94, p<0.01) and NRG-3GS (R2=0.97, p<0.01) models (Figure 1). This tight correlation of VAF was illustrated in hCD45+cells from xenografts transplanted with CMML, aCML or sAML cells. The majority of mutations detected were those in epigenetic regulator genes, such as ASXL1, EZH2 and TET2. No significant difference in VAF was observed between CD34+and CD34- compartments within the hCD45+cells. Additional samples, including specimens from patients with the related myeloproliferative neoplasm, chronic neutrophilic leukemia (CNL) are being analyzed and will be presented. These findings demonstrate the utility of SRG-W41-3GS as well as NRG-3GS as receptive hosts of primary human MDS/MPN cells with genetic evidence of their growth in these mice closely recapitulating the mutational profiles of the transplanted cells. These new strains may facilitate the development of functional screening and pre-clinical testing of novel therapeutic strategies for a range of human MDS/MPN and related myeloid disorders. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

Hypomethylation Therapy of Decitabine in Patients with Myelodysplastic Syndromes (MDS) Induces Apoptosis and Reduces Proliferation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 371-371
Author(s):  
I. Jilani ◽  
H. Kantarjian ◽  
M. Gorre ◽  
J.-P. Issa ◽  
J. Bennett ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Annexin V ◽  
Wilcoxon Test ◽  
Brdu Incorporation ◽  
Direct Result ◽  
Multiparameter Flow Cytometry ◽  
Aberrant Methylation ◽  
Mitochondrial Potential ◽  
Cd34 Cells ◽  
Significant Difference

Abstract Background: MDS is characterized by cytopenias believed to be the direct result of increased apoptosis of the hematopoietic cells in the bone marrow despite an increase in proliferation. Aberrant methylation resulting in leukemogenesis is frequent in MDS and is a potential target for pharmacologic therapy. Decitabine (Dacogen™; DAC), which has been shown to be effective in treating patients with MDS (Saba et al, Kantarjian et al), indirectly depletes methylcytosine, resulting in hypomethylation of target genes. However, it is not known whether this correction of methylation leads to reducing the apoptosis, allowing normal cells to grow, or by increasing the apoptosis killing off the tumor cells. Methods: We studied apoptosis and proliferation in patients with MDS treated on a randomized protocol to receive either DAC or best supportive care (SC). Apoptosis as measured by annexin V and mitochondrial potential was studied in various subpopulations of cells using multiparameter flow cytometry. Proliferation was also measured in a similar fashion using BrdU incorporation. Bone marrow (BM) and peripheral blood (PB) samples were collected from patients at baseline and at various times on therapy. Results: At baseline, the DAC group showed no significant difference in apoptosis, proliferation or percent of CD34+ cells from the SC group in BM (n = 39 and 33 patients, respectively) or PB (n = 51 and 50, respectively). After three months on treatment a significant increase in apoptosis (annexin V) in CD34+ cells was noted in the DAC arm but not in the SC arm (Wilcoxon test, P=0.01). Similar results were obtained when disturbance in mitochondrial potential was measured in the blast population (P=0.02). Interestingly, a similar increase in apoptosis was observed in CD8+ cells in the DAC arm (P=0.02). The increase in apoptosis was augmented with additional courses of DAC therapy. A greater reduction in proliferation (BrdU incorporation) in the CD34+ cells in the decitabine arm compared to SC (P=0.01) was also observed. Those DAC-treated patients who had a higher proliferation rate (BrdU incorporation) at diagnosis were more likely to achieve a CR than those with low level of proliferation (P=0.03). Conclusions: Decitabine therapy in patients with MDS leads to hypomethylation, but the net effects include high levels of apoptosis and the death of the neoplastic cells and a complimentary reduction of proliferation of the leukemic cells.

Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4808-4808
Author(s):  
Young-Ho Lee ◽  
Young-hee Kwon ◽  
Kyoujung Hwang ◽  
Hyunju Jun ◽  
Byungbae Park ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Conflicts Of Interest ◽  
T Cell Subsets ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Stromal Cell Derived Factor ◽  
And Control ◽  
Cellular Components

Abstract Abstract 4808 Background: It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC). Methods: The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured. Results: The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001). Conclusions: Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application. Disclosures: No relevant conflicts of interest to declare.

Abnormalities of Bone Marrow Immune Micro-Environment in Patients with Immune Thrombocytopenia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3464-3464
Author(s):  
Yang Song ◽  
Yu-tong Wang ◽  
Xiao-jun Huang ◽  
Yuan Kong
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Endothelial Cells ◽  
Th17 Cells ◽  
Immune Thrombocytopenia ◽  
Perivascular Cells ◽  
Platelet Production ◽  
Cd34 Cells ◽  
Significant Difference

Abstract Background: Immune thrombocytopenia (ITP) is an immune-mediated disease that is characterized by excessive platelet destruction and decreased platelet production. Although antiplatelet antibodies are considered as the primary immunologic defect in ITP, dysfunctional cellular immunity is also important in the pathophysiology of ITP. The current publications have observed excessive activation and proliferation of platelet auto-antigen-reactive CTLs, production abnormal Th cells, abnormal numbers and function of Tregs in peripheral blood of ITP, but no one focus on the bone marrow (BM) micro-environment in ITP patients. Many cell types including osteoblastic, perivascular, endothelial cells, and various mature immune cells contribute to the BM micro-environment. We have recently reported that the impaired BM vascular micro-environment may affect the thrombopoiesis of CD34+ cells by disrupting the interaction between megakaryocytes and BM endothelial cells (BMECs), resulting in the delayed platelet engraftment in allotransplant patients with prolonged isolated thrombocytopenia (Kong Y, et al. Biol Blood Marrow Transplant. 2014; 20:1190-1197). In mice model, the cross-talk between megakaryocytes and BMECs in BM vascular micro-environment regulates the megakaryocyte maturation and thrombopoiesis. Therefore, we hypothesized that the abnormal BM vascular micro-environment and immune micro-environment may operate in the occurrence of ITP. Aims: To investigate whether abnormal BM vascular and immune micro-environment are involved in ITP patients. Methods: The compartments of BM immune micro-environment were analyzed by flow cytometry in 26 untreated ITP patients and 26 healthy donors (HD). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+ CD8- IFN-gama+, CD3+ CD8- IFN-gama+, CD3+ CD8+ IL4+, CD3+ CD8+ IL-4+, CD3+ CD8- IL17A+ and CD3+ CD4+ CD25+ Foxp3+, respectively. The BMECs and perivascular cells, acting as key elements of vascular micro-environment, were identified as CD45- CD34+ VEGFR2+ and CD45- CD34- CD146+, respectively. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) using rabbit anti-human CD34 and CD146 primary antibodies were performed on each BM trephine biopsies (BMB) derived from the patients and controls. Results: The proportion of Th1 cells and Tc1 cells among the bone marrow mononuclear cells (BMMNCs) was significantly increased in ITP patients compared to HD (27.7% ± 11.6% vs. 16.3% ± 7.7%, P<0.001; 39.8%±17.7% vs. 24.1%±11.8%, P<0.005), whereas there was no significant difference in the percentages of Th2 and Tc2 cells. In addition, the proportion of Th17 cells in ITP patients was remarkable higher than HD (3.2%±0.51%1.5%vs 1.7%±1.0%, P<0.0001). We also found the significantly decreased percentage of Treg in ITP patients compared to HD (2.5%±2.0% vs 3.7%±2.6%, P<0.001). However, the frequency of CD34+ cells as well as BMECs and perivascular cells were similar in BM between the ITP patients and HD. Consistent with our flow cytometry data, histological analysis of the recipient BMBs in situ showed no significant differences in CD34-positive BMECs and CD146-positive perivascular cells between ITP patients and HD. Summary/Conclusion: The BM CD34+ cells and vascular micro-environment were normal in ITP patients. However, the abnormal BM immune micro-environment, including the excessive polarization of Th1, Tc1 and Th17 cells and a remarkable decrease of Treg cells were observed in ITP patients. Our data indicated that the desregulated T cells responses in BM may abrogate the thrombopoiesis through the impaired megakaryocytes maturation and decreased platelet production, and eventually contributing to the occurrence of ITP. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

scholarly journals Immunoexpression of CD34, CD117, and p53 in Hypocellular Bone Marrow Disorders

2021 ◽  
Author(s):  
Pooja Sharma ◽  
Anshu Palta ◽  
Anita Tahlan ◽  
Manveen Kaur ◽  
Ram Singh
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
The Other ◽  
Immunohistochemical Expression ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Blast Count ◽  
Bone Marrow Disorders ◽  
Acute Myeloid

Abstract Objectives Hypocellular bone marrow (BM) disorders comprise heterogeneous entities associated with peripheral cytopenias and decreased production of hematopoietic cells in BM. This study was undertaken to analyze immunohistochemical expression of CD34, CD117, and p53 in morphologically diagnosed patients of hypocellular BM (aplastic anemia [AA], hypocellular myelodysplastic syndrome [h-MDS], and hypocellular acute myeloid leukemia [h-AML]). Materials and Methods BM specimens were obtained from patients presenting with pancytopenia/bicytopenia. On 30 patients diagnosed as hypocellular BM, immunohistochemistry (IHC) for CD34, CD117, and p53 was performed. Results BM cellularity was < 30% in all (100%) patients. Blast count was increased in h-MDS and h-AML. Features of dysplasia were noted in six (20%) patients. Out of these, three patients were diagnosed as h-MDS having bilineage/trilineage dysplasia, and the other three patients were of AA (11.5% patients) displaying only dyserythropoiesis. On IHC, percentage of BM CD34+ cells was increased in h-MDS+ h-AML (3.87 ± 0.86) as compared with AA (0.19 ± 0.15) and controls (0.81 ± 0.21), p = 0.01. Percentage of BM p53+ cells was also increased in h-MDS+ h-AML (2.9 ± 2.07) as compared with AA and controls, which did not show any p53+ cells, p = 0.0. No statistically significant difference was observed in the expression of CD117 in h-MDS+ h-AML (4.95 ± 3.40) compared with AA (4.49 ± 1.07), p = 0.99. Conclusion The study demonstrates the usefulness of CD34 and p53 immunoexpression as an important ancillary method in distinguishing various hypocellular BM disorders, especially h-MDS and AA. However, the role of CD117 remains unclear and needs to be evaluated further by larger studies.

Erythropoiesis Is Highly Stimulated in CD34+ Cells in Low-Risk Myelodysplastic Syndromes (MDS) with an Improper Mitochondrial Function.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 473-473
Author(s):  
Ramin Tehranchi ◽  
Rosangela Invernizzi ◽  
Boris Zhivotovsky ◽  
Bengt Fadeel ◽  
Ann-Mari Forsblom ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Erythroid Differentiation ◽  
Low Risk ◽  
Refractory Anemia ◽  
Continuous Increase ◽  
Atp Production ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Cyt C

Abstract The apoptosis of early erythroblasts from patients with low-risk MDS, refractory anemia (RA) and RA with ringed sideroblasts (RARS), is mediated through a constitutive cytochrome c (cyt c) release from mitochondria (Tehranchi etal, 2003). Moreover, mature erythroblasts in RARS, but not in RA, show mitochondrial accumulation of aberrant ferritin (MtF) (Cazzola etal, 2003). This study aimed at further describing the pathophysiology of ineffective hematopoiesis in low-risk MDS, by studying cyt c release and MtF expression during erythroid differentiation and mitochondria ATP production in MDS bone marrow cells. We assessed freshly isolated CD34+ cells and day 4–14 erythroblasts from RARS, RA and normal bone marrow (NBM). CD34+ cells from all individuals were negative for MtF. NBM showed only few positive cells (0–4%, d4–14), and RA erythroblasts a median of 3% (0–8%) MtF+ cells. RARS erythroblasts, on the contrary, showed an early increase in MtF+ cells and a continuous increase during the culture period (d4=10%, d7=17%, d14=19%). There was a positive correlation between MtF expression and cyt c at day 14 ( r2=0.8). There was no significant difference in mitochondria ATP production between RARS, RA and NBM (all complexes or cyt -dependent complex IV). We found a significant over-expression (mRNA) of the pro-apoptotic genes for cyt c, Bid and Bax at day 0. Moreover, genes involved in erythroid differentiation were significantly up-regulated in MDS CD34+ cells: 6-fold for GATA-1 and 23-fold for β-globin; p&lt;,0005 for both. GATA-1 and β-globin expression increased during normal erythroid maturation, but in MDS erythroblasts GATA-1 declined and β-globin showed only a weak increase. Comparing RARS with RA, the former showed both higher expression of the β-globin and GATA-1 genes, and a higher degree of cyt c release and MtF expression. This indicates that the cellular abnormalities leading to erythroid apoptosis as well as efforts to compensate for these defects are present at the stem cell level in RARS. G-CSF that reduces cyt c release in MDS erythroblasts (RARS&gt;RA) showed no effect at all on ATP production or cyt c mRNA. Moreover, G-CSF tended to increase MtF expression in some RARS erythroblast cultures, indicating that it allows survival of pro-apoptotic MDS erythroblasts rather than addressing the cause of apoptosis. In conclusion, the aberrant MtF expression of RARS erythroblasts occurs at a very early stage of erythroid differentiation and is paralleled by an up-regulation of genes involved in erythroid differentiation. Alternative mechanisms may be involved in RA pathogenesis, since these cells show cyt c release but only moderate MtF expression, and less gene up-regulation.

Recovery of Viable CD34+ Cells from Cryopreserved Haemopoietic Stem Cell Products.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5018-5018
Author(s):  
Mary M. Sartor ◽  
Frances Garvin ◽  
Vicki Antonenas ◽  
Melina Webb ◽  
Kenneth F. Bradstock
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Count ◽  
Adult Stem Cell ◽  
Autologous Bone Marrow ◽  
Freeze Thaw ◽  
Cd34 Cells ◽  
Significant Difference ◽  
The Mean ◽  
Autologous Bone

Abstract The recovery of viable CD34+ cells reinfused into patients at the time of autologous or allogeneic transplantation is clinically an important variable, which can determine graft success or failure. In this study we analyse the recovery of viable CD34+ cells /kg pre and post cryopreservation on a total of 86 autologous stem cell products from adult and paediatric patients as well as 4 cryopreserved stem cell products from allogeneic donors. CD34 enumeration was performed on all samples pre and post cryopreservation using a novel in-house no-lyse CD34 assay (previously described ASH 2003 abstract no.1685). Cells were labelled with CD45, CD34 and 7AAD in TRUCOUNT tubes using a modified single platform ISHAGE protocol. The absolute number of viable CD34 + cells per Kg was determined. For the 77 PBSC harvest samples the mean viable CD34+ cell count was 6.0 x10^6/Kg (range 0.3 – 25.2 x 10^6/Kg) before freezing. For post thaw samples the mean viable CD34+ cell count was 5.5 x 10^6/Kg (range 0.2 – 24.6 x 10^6/Kg). The median recovery was 95% (range 48–124%). This represents a median loss post freeze/thaw of 5%. Further analysis showed a median recovery of 90% for NHL (range 48–119%, n=34), 87% for MM (range 56–115%, n=12), 92.5% for acute leukaemia (range 71–124% n=8) and 97% for non-hematological malignancies (range 50–120% n=21). There was no significant difference in the recovery of viable CD34+ cells within the four groups of malignancies (p>0.17 for all groups tested). Similarly, autologous bone marrow collections (n=9) also showed a good recovery of viable CD34+ cells post thaw. The median viable CD34+ cell count was 8.1x10^6 /Kg (range 0.6–30.3x10^6/kg) pre-cryopreservation, compared to a median viable cell count of 6.5 x10^6/Kg CD34+ cells (range 0.6–26x10^6/Kg) post thaw, this represents a median recovery of 90% viable CD34+cells from autologous bone marrow collections. There was no significant difference in the recovery of viable CD34+ cells from autologous PBSC harvests and autologous bone marrow collections (p=0.169). We also compared the recovery of viable CD34+ cells post thaw between adult and pediatric stem cells collections. The median recovery of viable CD34+ cells from 56 adult stem cell products post thaw was 91% (range 48–120%), compared to a median recovery of 96.5% (range 50–124%) viable CD34+ cells from 30 pediatric stem cell products (p=0.06). Interestingly the greatest loss occurred in allogeneic donors, where viable CD34+ counts on fresh samples averaged 5.7 x 10^6/Kg (range 3.1–11.8 x 10^6/Kg, n=4), whereas post freeze/thaw averaged 2.2 x 10^6/Kg (range 1.2–3.3 x 10^6/Kg). Representing a mean loss of 58% of CD34+ cells. Twenty-nine patients were transplanted with a median number of 3.8x10^6 viable CD34+ cells per Kg (range 1.8–18.4x10^6/Kg), The median time to neutrophil and platelet engraftment was 12 days (range 10–18) and 14 days (range 8–65) respectively. Assaying the viability of CD34+ cells post cryopreservation may identify patients at risk of poor haematological recovery that could benefit from further stem cell collections.

Apoptosis-Independent Activation of Caspase-3 in CD34–Positive (CD34+) Mobilized Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2305-2305
Author(s):  
Karine Augeul-Meunier ◽  
Carine Crampé ◽  
Philippe Farce ◽  
Christiane Mounier ◽  
Denis Guyotat ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Caspase 3 ◽  
Normal Bone Marrow ◽  
Hematopoietic Stem ◽  
Normal Bone ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Mobilized Peripheral Blood

Abstract G-CSF mobilized peripheral blood CD34+ cells are now the preferred and major source of hematopoietic stem and progenitor cells harvested for both autologous and allogeneic transplantation. Several mechanisms, like SDF-1/CXCR4 interactions or degradation of adhesion molecules by proteolytic environnement, are involved in the mobilization process. However this phenomenon is still partially understood. Gene expression analysis has identified an overexpression of the caspase-3 gene in CD34+ mobilized cells, compared to CD34+ from normal bone marrow. Caspase-3 is the main effector of the terminal phase of apoptosis. However recent studies have provided evidence of its implication in non apoptotic cellular processes, such as differentiation, migration and cytoskeleton modelling. We evaluated by multicolour flow cytometry the expression of activated caspase-3 in G-CSF mobilized CD34+/CD45+ cells from blood (n=16), and from apheresis products (n=10). CD34+/CD45+ cells from normal bone marrow (n=4) served as control. Caspase-3 activity on fluorescent substrate (PhiPhiLux method) and apoptosis (Annexin V assay) were also evaluated. Finally we analysed the expression of anti apoptotic proteins Bcl-2, Bcl-Xl, and of Heat Shock Proteins HSP27, HSP70 and HSP90 in the same cell population. There was no significant difference for apoptosis between mobilized and bone marrow CD34+ cells (26% versus 33% apoptotic cells). Activated caspase-3 levels were significantly higher in mobilized CD34+ cells (mean fluorescence intensity 3.64 fold higher). This was consistent with cleavage of caspase-3 substrate observed in mobilized cells, but not in bone marrow CD34+ cells. An increased expression of HSP90 (of which caspase-3 is a client protein) was observed in peripheral CD34+ cells, but there was no variation of BCl-2 and Bcl-Xl expression. Our results show an activation of caspase-3 in the mobilized peripheral blood CD34+ cells, which appears to be independent of apoptosis induction. The role of this activation and possible control by HSPs warrants further analysis to establish its relationship with mobilization mechanisms.

PDGFR-Mediated Cytomegalovirus Infection of Megakaryocytes Induces Thrombocytopenia Via Demethylation of AML1 and IEX-1 in the TPO/c-Mpl Signaling Pathway Following Allo-HSCT

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3937-3937
Author(s):  
Xiao-Hui Zhang ◽  
Feng Fei-er ◽  
Qian-ming Wang ◽  
Xiao-lu Zhu ◽  
Lan-ping Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Hcmv Infection ◽  
Treated Group ◽  
In Vitro Studies ◽  
Significant Difference

Abstract Introduction: Human cytomegalovirus (HCMV) infection is a common complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is associated with high morbidity and mortality. Thrombocytopenia is one of the major hematological complications of HCMV infection. Possible causes include direct HCMV injury to hematopoietic progenitor cells and the microenvironment, as well as HCMV-related immune thrombocytopenia. Previous in vitro studies demonstrate that HCMV could directly infect megakaryocytes(MKs) and their progenitors, resulting in decreased CFU-MK and increased apoptosis, but the underlying mechanisms remain uncertain. It remains unknown whether HCMV can directly target MKs in vivo, how MK function changes after infection, why HCMV selectively infects certain patients and what inhibits MK maturation and results in apoptosis. It has been reported that patients with HCMV-related thrombocytopenia showed poor response to rhTPO, implying blockage of the TPO/c-Mpl signaling pathway. Our previous research indicated that PDGFR+CXCR4lowCCR5lowMKs are correlated with HCMV infection.We hypothesized that PDGFR+CXCR4lowCCR5lowMKs are more susceptible to HCMV infection. HCMV could directly target MKs both in vitro and in vivo, resulting in increased apoptosis and decreased MK ploidy. HCMV infection could possibly disturb the downstream TPO/c-Mpl signaling pathway, thereby inhibiting MK differentiation and maturation. Methods: We collected bone marrow from HCMV DNAemia patients post allo-HSCT for in vivo study. Transmission electron microscopy(TEM) was used to detect HCMV particles inside MKs. MKs were identified as CD41+vWF+cells by flow cytometry(FCM). To analyze the susceptibility of MKs to HCMV, expression levels of PDGFR, αvβ3, TLR2, CCR5 and CXCR4 in different groups were tested. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. We also measured c-Mpl expression in MKs.In vitro study, we used plasma from HCMV-infected patients post allo-HSCT to infect MKs cultured from bone marrow CD34+ cells. We validated cell susceptibility with the same markers used in vivo. Next, inhibitors of the positive markers were co-cultured with MKs. We analyzed pp65 expression in the inhibitor-treated group and control group to explore potential prevention of HCMV infection. We investigated AML1 and IEX-1 in the downstream TPO/c-Mpl signaling pathway by PCR and Western Blot. We used bisulfite sequencing PCR (BSP) to study the methylation status in different gene expression profiles of AML1 and IEX-1. 5-ara-dC is a type of DNA methylation inhibitor. After incubation with MKs, we analyzed changes in gene expression and MKs function. Results: Using TEM, we managed to find HCMV particles in MKs from HCMV-infected patient bone marrow samples. The proportion of apoptosis markedly increased compared with HCMV-negative MKs, whereas the mean ploidy slightly decreased. C-Mpl expression showed no significant difference between the two groups. Pp65 positive cells showed elevated expression in PDGFR and reduced expression in CXCR4 and CCR5. In vitro studies revealed similar results. After treating with the PDGFR inhibitor IMC-3G3, the pp65 positive cell population was slightly decreased, but the Gleevec-treated group showed no difference. We found a decrease in both IEX-1 and AML1 on both the molecular and protein levels. Both gene promoters were hypermethylated in the HCMV-infected group. After demethylation with 5-ara-dC, IEX-1 and AML1expression levels were both up-regulated, and cell apoptosis was reduced. Conclusion: (1)HCMV inhibited megakaryocytic differentiation and maturation and reduced MKs polyploidy both in vivo and in vitro. (2)MKs positive for PDGFR and low in CXCR4 and CCR5 were more susceptible to HCMV infection. The PDGFR inhibitor IMC-3G3 protected MKs from HCMV infection. (3)The mechanism of HCMV-associated thrombocytopenia may be a disturbance of the TPO/c-Mpl signaling pathway in MKs through hypermethylation of the AML1 and IEX-1 promoters. Demethylation with 5-ara-dC could reverse cell apoptosis. Therefore, we illustrated the possible mechanism of HCMV-induced thrombocytopenia, highlighting new insights for future potential therapeutic approaches. Disclosures No relevant conflicts of interest to declare.

In vivo efficacy of anti-MPL agonist antibody in promoting primary human hematopoietic cells

Blood ◽  
2009 ◽  
Vol 113 (10) ◽  
pp. 2213-2216 ◽  
Author(s):  
Masayuki Kai ◽  
Tetsuya Hagiwara ◽  
Chie Emuta ◽  
Yukiko Chisaka ◽  
Kumi Tsuruhata ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Therapeutic Potential ◽  
Hematopoietic Cells ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Total Body

Abstract In a previous study, we generated novel antithrombopoietin receptor agonist antibodies as therapeutic candidates. In this report, we investigated the in vivo effects of one of these antibodies, MA01G4344U, on primary human hematopoietic cells using xenotransplantation. NOD/Shi-scid, IL-2Rγnull (NOG) mice were pretreated by total-body irradiation and received a transplant of human cord blood–derived CD34+ cells. Weekly intraperitoneal injection of MA01G4344U (100 μg/mouse per week) or Peg-rhMGDF (5 μg/mouse per week) or phosphate-buffered saline (PBS) was performed. Human cells in peripheral blood were analyzed by flow cytometry and bone marrow cells were analyzed by flow cytometry and colony assay. MA01G4344U successfully increased the number of human CD41+ platelets and human CD45+ cells in peripheral blood. In the bone marrow, MA01G4344U increased the number of human CD45+/CD34+ cells, which resulted in more multilineage progenitor cells. The efficacy of MA01G4344U in promoting primary human hematopoietic cells in vivo suggests its therapeutic potential for thrombocytopenic and pancytopenic disorders.

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