scholarly journals Co-administration of human MSC overexpressing HIF-1α increases human CD34+ cell engraftment in vivo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Silvia Preciado ◽  
Mª Salomé Sirerol-Piquer ◽  
Sandra Muntión ◽  
Lika Osugui ◽  
Gerardo J. Martí-Chillón ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Cord Blood ◽  
Cell Transplantation ◽  
Attractive Potential ◽  
Human Cord Blood ◽  
Human Cd34 ◽  
Cell Engraftment

Abstract Background Poor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34+ human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo. Methods Human MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34+ cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34+ cells. In addition, CD34+ cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34+ cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice. Results We did not observe significant differences in viability, cell cycle and ROS expression between CD34+ cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34+ cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34+ cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34+ cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34+ co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively). Conclusions Co-transplantation of human CD34+ cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche.

siRNA-Induced Transient Silencing of PTEN Expression Enhances Human Hematopoietic Cell Engraftment in NOD/SCID/γcnull Mice and Increases Gene Transduction Efficiency.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2329-2329
Author(s):  
Inho Kim ◽  
Andre Larochelle ◽  
Yoo-Jin Kim ◽  
Cynthia E. Dunbar
Keyword(s):  
Cell Cycle ◽  
Gene Therapy ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Pten Expression ◽  
Retroviral Transduction ◽  
Human Cd34 ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
The Impact

Abstract PTEN (phosphatase and tensin homologue) has been implicated as a regulator of murine hematopoietic stem cell (HSC) self-renewal. In mice, PTEN deletion initially results in a transient expansion of HSC numbers, but the HSC pool subsequently becomes depleted. Therefore, constitutive down-modulation of PTEN would not be desirable for stem cell expansion, however transient depletion of PTEN activity could be beneficial for transplantation and gene therapy applications. Little is understood regarding the role of PTEN in human HSC regulation. We studied the impact of transient inhibition of PTEN expression in human CD34+ cells on their cell cycle profile, their susceptibility to retroviral transduction, their proliferative potential in vitro, and their ability to repopulate NOD/ SCID/γcnull mice. Human G-CSF-mobilized CD34+ cells were transfected with PTEN siRNA (PTEN1 or PTEN2) or control siRNA. Forty-eight hours later PTEN expression was inhibited by 73–100% (PTEN1) and 64–97% (PTEN2) compared to CD34+ cells transfected with control siRNA as measured by real-time RT-PCR. Reduced PTEN protein levels were confirmed by Western blot. Compared to control siRNA treated CD34+ cells, PTEN-silenced CD34+ cells showed a significant decrease in the proportion of cells in the G0 phase of the cell cycle (PTEN1: 10.9%; PTEN2: 13.0%; control: 19.5%; p < 0.05) and a concomitant increase in the proportion of cells in the S + G2/M phase of the cell cycle (PTEN1: 42.1%; PTEN2: 42.4%; control: 37.2%; p < 0.05). We hypothesized that the increased proportion of cycling CD34+ cells may enhance their susceptibility to retroviral transduction for gene therapy applications. Human CD34+ cells were nucleofected with PTEN or control siRNA, cultured for 2 days with cytokines and transduced for 48 hours with MND.MFG.YFP c10 retroviral vectors (MOI=0.3) on fibronectin-coated plates. Transduction efficiencies in the bulk CD34+ cells transfected with PTEN1 siRNA and PTEN2 siRNAs were significantly higher compared with CD34+ cells transfected with a control siRNA as determined by detection of YFP by flow cytometry. The average percentage of YFP+ cells from eight independent transductions were 41.7% (PTEN1, p=0.0001), 38.9% (PTEN2, p=0.003), compared with the control siRNA treated group (29.4%). We next tested whether transient inhibition of PTEN expression could result in improved engraftment of primitive human cells capable of repopulating nonobese diabetic/ severe combined immune-deficient-interleukin-2R−/− (NOD/SCID/γcnull) mice. A total of 25 NOD/SCID/γcnull mice were transplanted with PTEN or control siRNA treated CD34+ cells immediately after nucleofection (PTEN1 n=10; PTEN2 n=5; control n=10) and 9 additional mice were transplanted with untreated human CD34+ cells. Mice transplanted with PTEN1 siRNA- or PTEN2 siRNA-treated CD34+ cells had increased percentages of human cell engraftment as determined by flow cytometry for human CD45 compared to mice transplanted with control siRNA-treated CD34+ cells or untreated CD34+ cells (PTEN1: 37.6%; PTEN2: 38.1%; control: 19.7%; untreated: 19.2%, p = 0.03). NOD/ SCID/γcnull mice transplanted with PTEN1 and PTEN2 siRNA-treated CD34+ cells showed no statistically significant differences in human lymphoid (CD3+, CD20+) or myeloid (CD15+) differentiation compared to control mice but a trend favoring an increased representation of myeloid (CD15+) cells was noted in PTEN1-treated mice (PTEN1: 41.7%; control: 18.8%, p = 0.06). None of these mice developed myeloproliferative disorders or leukemias. Overall, these data suggest that PTEN plays an important role in governing transitions between the quiescent and activated states of primitive human hematopoietic cells, and the transient inhibition of PTEN expression in these cells results in improved retroviral transduction efficiencies and increased engraftment. This approach may find clinical applications in gene therapy for inherited disorders and in adult cord blood stem cell transplantation where the number of HSC is limited.

The Intracoelomic Route: A New Approach for in utero Human Cord Blood Stem Cell Transplantation

2003 ◽  
Vol 19 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Giuseppe Noia ◽  
Luca Pierelli ◽  
Giuseppina Bonanno ◽  
Giovanni Monego ◽  
Alessandro Perillo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cord Blood ◽  
Cell Transplantation ◽  
In Utero ◽  
Blood Stem Cell ◽  
Human Cord Blood ◽  
New Approach ◽  
Cord Blood Stem Cell ◽  
Blood Stem Cell Transplantation

High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle

Blood ◽  
2009 ◽  
Vol 113 (12) ◽  
pp. 2661-2672 ◽  
Author(s):  
Alex J. Tipping ◽  
Cristina Pina ◽  
Anders Castor ◽  
Dengli Hong ◽  
Neil P. Rodrigues ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cord Blood ◽  
Cell Function ◽  
Ki 67 ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cord Blood Cells ◽  
Low Efficiency

Abstract Evidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here, we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (G0 residency) of murine and human hematopoietic cells. In human cord blood, quiescent fractions (CD34+CD38−HoechstloPyronin Ylo) express more GATA-2 than cycling counterparts. Enforcing GATA-2 expression increased quiescence of cord blood cells, reducing proliferation and performance in long-term culture-initiating cell and colony-forming cell (CFC) assays. Gene expression analysis places GATA-2 upstream of the quiescence regulator MEF, but enforcing MEF expression does not prevent GATA-2–conferred quiescence, suggesting additional regulators are involved. Although known quiescence regulators p21CIP1 and p27KIP1 do not appear to be responsible, enforcing GATA-2 reduced expression of regulators of cell cycle such as CCND3, CDK4, and CDK6. Enforcing GATA-2 inhibited human hematopoiesis in vivo: cells with highest exogenous expression (GATA-2hi) failed to contribute to hematopoiesis in nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice, whereas GATA-2lo cells contributed with delayed kinetics and low efficiency, with reduced expression of Ki-67. Thus, GATA-2 activity inhibits cell cycle in vitro and in vivo, highlighting GATA-2 as a molecular entry point into the transcriptional program regulating quiescence in human hematopoietic stem and progenitor cells.

Correlation between Hematopoietic Stem Cell Engraftment Assay Results and Bone Marrow Biopsy, Flow Cytometry, and Cytogenetics Findings.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4961-4961
Author(s):  
Edward G. Weir ◽  
Kathleen Murphy ◽  
Denise Batista ◽  
Constance A. Griffin ◽  
Michael J. Borowitz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Host Cells ◽  
Fish Analysis ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract Hematopoietic stem cell transplantation following induction chemotherapy is an increasingly successful therapeutic option for patients with leukemia or lymphoma. The use of molecular assays in post-transplant patients has become the standard in evaluating these patients for evidence of engraftment or early recurrence of disease. The detection of residual host cells in the bone marrow (BM) or peripheral blood (PB) following stem cell transplantation often influences subsequent clinical management. The aim of our study is to determine the extent of correlation between the results of PCR-based stem cell engraftment (SCE) assays and BM biopsy (BMBx), multiparameter flow cytometry (FC) and cytogenetics findings in patients who have undergone stem cell transplantation as therapy for hematolymphoid malignancies. We retrospectively reviewed the results of 1103 serial SCE assays performed at The Johns Hopkins Hospital, and 596 of these had temporally corresponding BMBx, FC and/or cytogenetic analysis. Concordance between the results of SCE analysis and those of the latter assays was defined as the detection of similar host/donor compositions. While some cases demonstrated clear discordance between the results, a subset showed an equivocal correlation due to the unclear significance of <5% host DNA by SCE analysis. Of 318 SCE assays with concurrent BMBx, 167(52%) showed concordant results, 104(33%) showed discordant results, and 47(15%) demonstrated an equivocal correlation. Of 221 SCE assays with concurrent FC, 111(50%) showed concordant results, 73(33%) showed discordant results, and 37(17%) demonstrated an equivocal correlation. Additionally, SCE assays were performed on concurrent, paired BM and PB specimens in 168 patients. Concordant results were identified in 141(84%) pairs. Of the remaining 27 pairs, host DNA was detected in the PB of 16 cases in which the BM showed either donor only DNA (6 cases) or <5% host DNA (10 cases). Four cases showed <5% host DNA in the PB and chimeric DNA in the BM. However, donor only DNA was detected in the PB in 7 cases that demonstrated a chimeric BM. Lastly, concurrent SCE analysis and XY FISH analysis was identified in 28 cases. Concordance between these two assays was observed in 24 (86%) cases, whereas one (3%) case was discordant and 3 (11%) cases showed an equivocal correlation. In conclusion, both BMBx and FC show similar but weak correlations to SCE analysis. In contrast, XY FISH analysis demonstrates a strong correlation to SCE analysis. Furthermore, SCE analyses performed on paired PB and BM specimens show an overall good correlation. However, our data suggest that in a subset of cases, SCE analysis performed on PB may detect residual host DNA that is not detectable by SCE analysis of BM, possibly due to the heterogeneity of the marrow composition.

Lentivirally Transduced Human Cord Blood CD34+FLT3-ITD+ Cells Induce Murine Acute Leukemia in the NOD/SCID Transplantation Model.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2984-2984
Author(s):  
Pawan Kaur Bollinger ◽  
Doris Steinemann ◽  
Rama Krishna Kancha ◽  
Leticia Quantanilla-Fend ◽  
Stefan K Bohlander ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Leukemia ◽  
Cord Blood ◽  
Fold Increase ◽  
Bone Marrow Niche ◽  
Human Cord Blood ◽  
Human Cd34 ◽  
Cord Blood Cells

Abstract Abstract 2984 Targeting constitutively activated FLT3 (FLT3-ITD) by tyrosine kinase inhibition (TKI) in acute myeloid leukemia (AML) leads to clearance of blasts in the periphery but not in the bone marrow, suggesting a protective effect of the marrow niche on leukemic stem cells (LSC). We have previously shown that interaction of CD34+FLT3-ITD+ LSC with stromal niche cells mimicking the bone marrow environment specifically protects these cells from the effects of TKI and confers a growth advantage to FLT3-ITD+ leukemic stem/progenitor cells over normal ones (Parmar et al, Cancer Research 2011). To study human FLT3-ITD+ LSC in vivo in the context of the bone marrow niche, we aimed to establish a xenogeneic NOD/SCID mouse model of human FLT3-ITD+AML. Human CD34+ enriched cord blood cells were transduced with a pWPI lentivirus containing a VSV-pseudotyped SIN/LTR vector with eGFP and full length human FLT3 cDNA harboring a 30 bp length internal tandem duplication (FLT3-ITD) or empty vector control. Transduction efficiency ranged between 1–4.4% for FLT3-ITD and 1.3–18% for vector control. Sub-lethally irradiated NOD/SCID mice were then transplanted with 1 × 104 – 7 × 104 unselected or GFP-sorted CD34+FLT3-ITD+ cells. Acute leukemia developed in 7/9 animals after a median latency of 85 days (range 70–168), with involvement of peripheral blood, bone marrow, spleen and liver. Three mice developed acute lymphoblastic leukemia (ALL) whereas the remaining mice showed signs of AML. In contrast, mice receiving empty pWPI vector-transduced human CD34+ cord blood cells (n = 8) all remained healthy during the observation period of 28 weeks and, in 4/8 animals, normal human CD34+cells could be recovered from the bone marrow (human engraftment range 0.3–35.5%). Leukemic mice exhibited hepatomegaly and splenomegaly with an average 10-fold increase in spleen weight, 2-fold increase in spleen length and 2.7-fold increase in liver weight compared to control mice. In mice that developed ALL, lymph node enlargement was also noted. Whole bone marrow, spleen and liver cells from primary mice were re-transplanted and were able to reproduce acute leukemia in all secondary (n=10/10) and tertiary mice (n=11/11) with a median latency of 25 and 20 days, respectively (p<0.01). Surprisingly, detailed immunophenotypical and immunohistochemical analysis revealed all leukemias to be of murine origin. Leukemic cells stained positively for murine CD45.1 antigen but negatively for human CD45. However, a small population of human CD34+CD45+ cells (range 1–7%) was continuously detectable in the bone marrow of primary, secondary and tertiary transplanted leukemic mice. Accordingly, human FLT3-ITD was detectable by PCR specific for human FLT3 up to the third serial transplantation. Viral integration site analysis by LM-PCR on genomic DNA isolated from spleens of leukemic mice revealed lentiviral integration into the human genome, excluding the possibility of in vivo viral shuttling from human cord blood CD34+ cells to mouse hematopoietic stem cells. Moreover, multicolor fluorescent in situ hybridization (M-FISH) on metaphases generated from peripheral blood lymphocytes revealed only murine chromosomes, also ruling out the possibility of fusion between human and mouse cells. To further characterize these murine leukemias, we performed array CGH on murine spleen gDNA of four immunophenotypically different mice. All mice showed recurrent clonal chromosomal aberrations frequently found in AML. Surprisingly, we have no evidence for the presence of FLT3-ITD in these murine leukemias, suggesting that CD34+FLT3-ITD+ stem cells can trigger development of acute leukemia. We propose that leukemogenesis may mechanistically be related to the host microenvironment and that the bone marrow niche in NOD/SCID mice is susceptible to modulation by the FLT3-ITD oncogene. Disclosures: No relevant conflicts of interest to declare.

Targeting CD96 For Antibody Based Elimination Of Leukemic Stem Cells In AML: A New Strategy In Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3972-3972 ◽  
Author(s):  
Matthias Staudinger ◽  
Christian Kellner ◽  
Matthias Peipp ◽  
Natalie Schub ◽  
Andreas Humpe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Autologous Stem Cell Transplantation ◽  
Target Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Abstract Although the mortality of autologous stem cell transplantation in contrast to allogeneic is low, in AML patients the lack of immune surveillance as well as contamination of the transplant with residual leukemic stem cells (LSC) limits its use. Therefore, elimination of LSC by targeted therapy may represent a promising therapeutic approach. Recently, CD96 was identified as marker antigen on AML-LSC (Hosen et al., PNAS 104: 11008, 2007). Here, by addressing CD96 with magnetic cell sorting (MACS) or using antibody dependent cellular cytotoxicity (ADCC), new strategies for engineering autologous stem cell grafts or for in vivo targeting of residual AML stem cells are presented. To evaluate the efficacy of depletion of LSC by MACS technology, grafts containing hematopoietic stem cells were spiked with CD96 positive AML cells. Using biotinylated CD96 antibody TH111 raised in our laboratory in combination with anti-biotin-micro beads (Miltenyi Biotech, Bergisch Gladbach, Germany) up to a 1000-fold depletion of targeted cells was achieved. The viability, cell count and the potential of hematopoietic progenitor cells (HPC) to proliferate and differentiate were not affected by this procedure as documented by flow cytometry and colony forming assays. As residual LSC residing within the patient may also account for AML relapse after high-dose chemotherapy and subsequent SCT, eradication of AML stem cells in vivo is desirable. To target CD96+ AML-LSC by ADCC, chimeric antibodies containing wild type or affinity maturated variable regions in combination with an optimized human IgG1Fc were generated by recombinant DNA technologies. Both recombinant antibodies were expressed in Hek 293 cells enriched to homogeneity by affinity chromatography and analyzed for their functional properties. As shown by flow cytometry, the antigen binding affinity of the maturated antibody was enhanced (EC50 0.6 μg/ml vs. 2 μg/ml). Moreover, as analyzed in standard ADCC assays, NK cell mediated lytic properties against CD96-positive target cells were elevated (maximum lysis: 52%) using the affinity maturated chimeric CD96 antibody (EC50: 0.02 μg/ml vs. 0.15 μg/ml). Thus, this CD96 purging strategy avoids unwanted transplantation of AML-LSC and may help to revitalize autologous stem cell transplantation in this indication. Although, specific side effects by CD96 application will have to be considered, this may allow for an additional therapeutic avenue to eliminate in vivo residual AML-LSC in autologous as well as in allogeneic situations. Disclosures: No relevant conflicts of interest to declare.

HLA Class I Typing of Platelets by Multi-Colour Flow Cytometry - A New Powerful Tool for Assessing Survival, Activation Status and Activation Capacity of Transfused Platelets Vs. Autologous Platelets and for Determination of Stem Cell Engraftment After Allogeneic Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 22-22 ◽  
Author(s):  
Annette Vetlesen ◽  
Pål Andre Holme ◽  
Torstein Lyberg ◽  
Jens Kjeldsen-Kragh
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Hla Class I ◽  
Class I ◽  
Hla B27 ◽  
Cell Engraftment ◽  
Activation Capacity

Abstract Abstract 22 Studies of in vivo survival of transfused platelets (PLTs) are usually performed by tracing PLTs labelled with radioactive isotopes. The aim of the present work was to develop a flow cytometry-based method without involving manipulation of PLTs before transfusion, where differences in HLA class I molecules between donor and recipient might be used to trace transfused PLTs. Of 14 fluorochrome-conjugated HLA class I monoclonal antibodies (mAbs) from 7 suppliers only 3 (anti-HLA A2, anti-HLA A9 and anti-HLA B27) were found satisfactory for HLA class I typing of PLTs. As earlier studies have claimed the existence of a considerable exchange of HLA class I antigens between plasma and PLTs, a series of experiments were conducted to examine the exchange of HLA A2, A9 and B27 class I antigens during storage for 4 days. Three pairs of HLA +ve and HLA −ve PLT aphaeresis products were collected in autologous plasma for each of the specificities HLA A2, HLA A9 and HLA B27. Cell-free plasma was prepared from an equal number of HLA +ve and HLA −ve whole blood units with the same specificities. Plasma-free PLTs and cell-free plasma were mixed and stored in PLT storage bags at 22°C in the following combinations: HLA +ve PLTs in HLA −ve plasma, HLA +ve PLTs in HLA +ve plasma, HLA −ve PLTs in HLA −ve plasma and HLA −ve PLTs in HLA +ve plasma. Samples from each PLT concentrate-mix were transferred to sterile non-treated culture plates and stored on a flatbed mixer at 37°C in an atmosphere of 5% CO2 in humidified air. HLA class I surface expression was tested daily during storage. The difference in % between pos PLTs in neg plasma and pos PLTs in pos plasma was in the range of 0.8 – 1.2 at 22°C and 0.8 – 3.3 at 37°C for HLA A2; 3.3 – 1.7 at 22°C and 3.5 – 0 at 37°C for HLA A9, and 3.9 – 1.9 at 22°C and 0 – 2.1 at 37°C for HLA B27. Percent-wise difference between neg PLTs in pos plasma and neg PLTs in neg plasma was in the range of 6.9 – 10 at 22°C and 7.7 – 12.5 at 37°C for HLA A2; 4.5 – 0 at 22°C and 13.6 – 6.5 at 37°C for HLA A9, and 5.3 – 5.3 at 22°C and 5.6 – 4.8 at 37°C for HLA B27. These results indicate that the amounts of eluted vs. adsorbed HLA class I antigens are negligible at both 22°C and 37°C. Hence, this method was applied in a clinical setting to study in vivo survival of transfused PLTs. A patient was transfused with PLTs of non-self HLA class I types and tracing and testing of the transfused PLTs were performed by daily measuring of HLA class I surface expression by multi-colour flow cytometry. PLTs were identified by light scatter properties and expression of CD41. The anti-HLA class I mAbs were used to distinguish transfused PLTs from autologous PLTs. The activation status of transfused PLTs was determined by using anti-CD63. PLT activation capacity was further determined by examining the expression of CD63 on different PLT populations before and after stimulation with thrombin receptor agonist peptide (SFLLRN). In a 52 years old patient with AML undergoing allogeneic stem cell transplantation early stem cell engraftment could be detected by following the PLT production (see figure). After the transplantation the number of autologous HLA A2 +ve PLTs decreased gradually. From day 8 till day 10 there was a 50% decrease in total PLT count, while the number of HLA A2 +ve PLTs was low but stable. From day 10 and onwards a gradual increase of HLA A2 +ve PLTs was seen independently of transfusion. These results were interpreted as engraftment from day 8. The standard criteria for stem cell engraftment - a neutrophile count of > 0, 1 × 109/L - occurred 5 days later in this patient. It was also found that the transfused PLTs in the patient's circulation were not activated, evaluated by CD63 expression, and that the residual activation capacity of the transfused PLTs were reduced compared with the capacity of the autologous PLTs (40% up regulation of CD63 vs. 70%; as a comparison there was a 90% up regulation of CD63 in a healthy individual). In conclusion, HLA class I typing by flow cytometry represents a powerful tool for studies of transfused PLTs. The method can 1) be used to study the survival of 3 different populations of transfused PLTs individually, 2) give early evidence of stem cell engraftment after allogeneic stem cell transplantation, 3) be used to assess in vivo activation of transfused PLTs and 4) be used for functional studies of transfused vs. autologous PLTs, which to our knowledge, is the first time this has been possible. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Functional Studies of Ex Vivo Expanded Hematopoietic Stem Cells in Mice and Monkeys

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1899-1899
Author(s):  
Yu Zhang ◽  
Bin Shen ◽  
Meng Qin ◽  
Zhihua Ren ◽  
Xinxin Ding ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Autologous Transplantation ◽  
Hematopoietic Stem ◽  
Cynomolgus Monkeys ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Bone Morrow

Abstract Hematopoietic stem cell (HSC) transplantation has been widely applied for the treatment of malignant blood diseases. However, obtaining sufficient HLA-matched stem/progenitors for cell transplantation is an obstacle for clinical applications. We reported here that an optimal cytokine cocktail in a modified IMDM basal medium was developed that contained stem cell factor, Flt-3 ligand, thrombopoietin, interleukin 3, G-CSF and GM-CSF. Up to 7.3 folds of expanded CD34+ cells with 66.3% CD34+ of whole cells were obtained after 4 days' culture from human umbilical cord blood. Colony-forming unit (CFU) assays showed that expanded CD34+ cells retained the same renewal ability as the pre-expanded counterparts. To test the repopulating ability of the expanded CD34+ in vivo, sixteen NOD/SCID mice were divided to four groups and injected with saline (group 1), 0.4 million pre-expanded CD34+ cells (group 2), 0.4 million 4-day expanded CD34+ cells (group 3), and 2.9 million expanded CD34+ cells (group 4), respectively. Multi-lineage differentiations in the peripheral blood were assessed by flow cytometry with antibodies against a panel of human cell surface markers. In week 3, human CD34+ cells were decreased below 1% in groups 2 and 3, and 1.717%±0.65% in group 4. Whereas, human CD45+ was increased up to 3.831%±1.54%, 3.108%±1.18% and 10.408%±3.27% for groups 2, 3 and 4, respectively. The other human CD41+, CD71+ and CD15+ were also increased in groups 2-4. No expression of any human cell lineage markers was detected in group 1, indicating that expanded human CD34+ cells possessed the repopulating viability of HSCs in vivo. Furthermore, in week 12, the human CD34+ cells were re-isolated from the bone morrow of the mice (one mouse from each group). The isolated human CD34+ cells were again transfused into new NOD/SCID mice for the secondary transplantation. In week 6, human CD45+, CD15+ and CD19+ were observed from the bone morrow cells of sacrificed mice. On the other hand, human CD45+, CD15+ and CD19+ were also detectable in bone morrow cells for all remaining mice in week 24, suggesting that the expanded CD34+ cells could be successfully engrafted into mice in a long term. In addition, the cytokine cocktail was further evaluated for its safety and efficacy in primates. The CD34+ cells were isolated from the peripheral blood of cynomolgus monkeys and expanded for about 8 folds were obtained on day 9. Harvested CD34+ cells were transducted with the gene of green fluorescent protein (GFP). These cynomolgus monkeys (n=11) were administered with cyclophosphamide via intravenous injection at a dose of 50 mg/kg/day for two days. The myelo-suppressed monkeys were randomly divided into three groups as follows: a control group treated with saline (n=3), a group with autologous CD34- cells (n=3), and a group treated with GFP-labeled, expanded autologous CD34+ cells (n=5), respectively. After autologous transplantation, routine blood tests and flow cytometry analysis were performed to determine the proportion of GFP+ cells in the peripheral blood. The flow cytometry analysis revealed that the white blood cells (WBC), neutrophil (NEU) and platelets (PLT) in peripheral blood of cynomolgus monkeys were completely recovered to the normal levels on days 12, 11 and 10 post autologous transplantation of expended CD34+ cells, respectively. For the control groups, WBC, NEU and PLT returned to the normal on days 22, 22 and 12 for the saline treatment and on days 20, 20 and 12 for the CD34- group, respectively. Similarly, the lymphocytes of cynomolgus monkeys were recovered completely on day 20 post autologous CD34+ cell transplantation compared with the saline control (day 25) and the CD34- group (day 22). On day 30 after the autologous transplantation, the GFP+ ratio in CD45+ populations was around 2% in the peripheral blood. GFP+ cells (ranging from 1.8% to 4.1%) were also detected in bone morrow of cynomolgus monkeys. All primates transplanted with the expanded autologous CD34+ cells have survived for 18 months without any noticeable abnormalities. In conclusion, our results indicate that expanded CD34+ cells can be safely and efficiently used for repopulating stem cell compartment in mice and primates, underscoring the potential applications in the clinic. Furthermore, the results from successful autologous transplantation of cynomolgus CD34+ cells strongly suggest a possible application for personalized treatment of blood diseases. Disclosures Qin: Biopharmagen. corp: Employment. Ren:Biopharmagen corp: Employment.

Competitive Repopulation Assay for Human Stem Cell Engraftment in NOD-scid IL-2rγnul Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5215-5215
Author(s):  
Lisa J. Giassi ◽  
Joseph Laning ◽  
Kristen Biber ◽  
Amy Cuthbert ◽  
Lowry A. Phillip ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Animal Studies ◽  
Selective Advantage ◽  
Clinical Report ◽  
Cell Numbers ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Hematopoietic Chimerism

Abstract Umbilical cord blood (UCB) is a source of stem cells for hematopoietic transplantation (Laughlin, et al. NEJM2004; 351:2265) but application in adults is limited by marginal stem cell numbers. Combining cord blood units has shown promise in achieving engraftment (Barker, et al. Blood2002; 100:41a [abs #142]) without crossed immunologic rejection. Short-term hematopoiesis showed contributions from both cords, but long-term engraftment derived predominantly from just one cord. To study this process we have developed an in vivo pre-clinical competitive repopulation assay using NOD-scid IL-2rγnull mice that support high levels of human UCB engraftment (Shultz, et al, J. Immunol.2005, 174:6477). NOD-scid IL-2rγnull mice were transplanted with T-cell depleted UCB containing 3x104 HLA-A2+ CD34+ cells, 3x104 HLA-A2− CD34+ cells, or a 1:1 mixture (6x104 CD34+ cells). Human hematopoietic chimerism, measured by human CD45 and HLA-A2 expression, was quantified in bone marrow 6 and 12 weeks after transplantation. Variable mixed engraftment within individual as well as between 6 separate experiments was observed. At both 6 and 12 weeks, mixed contribution from both cords was achieved although rarely in a 1:1 ratio despite the infusion of equivalent CD34+ cell numbers. HLA-A2 expression did not provide a selective advantage. The complete dominance of one cord observed in the clinical report and in animal studies (Yahata, et al. Molecular Therapy2004; 10: 882) suggests an important role for the immune activity of UCB T-cells in this competitive process, a hypothesis we are currently testing. This model has a rapid turnaround and can test multiple permutations, providing a preclinical vehicle for characterizing optimal combinations of cord cells and for testing conditions that allow one cord to ultimately prevail. Legend: Engraftment of human CD45+ cells at 6 weeks. All bars represent single mice except for the HLA-A2+ group, which is the average (±SEM) of 3 mice. Legend: Engraftment of human CD45+ cells at 6 weeks. All bars represent single mice except for the HLA-A2+ group, which is the average (±SEM) of 3 mice.

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