Ex Vivo Cultured Megakaryocytes Express Functional Glycoprotein IIb-IIIa Receptors and Are Capable of Adenovirus-Mediated Transgene Expression

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4084-4092 ◽  
Author(s):  
Nauder Faraday ◽  
Jeffrey J. Rade ◽  
David C. Johns ◽  
Gopal Khetawat ◽  
Stephen J. Noga ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Blood Platelets ◽  
Green Fluorescence Protein ◽  
Hematopoietic Stem ◽  
Development Factor ◽  
Fibrinogen Binding ◽  
Activation Characteristic ◽  
Fluorescence Protein

Investigation of the molecular basis of megakaryocyte (MK) and platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence of pegylated recombinant human MK growth and development factor (PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as assessed by agonist inducible soluble fibrinogen and PAC1 binding. The specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa receptors were similar to those described for blood platelets. The reversibility and internalization of ligands bound to MK GPIIb-IIIa also shared similarities with those observed in platelets. Cultured MKs were transduced with an adenoviral vector encoding green fluorescence protein (GFP) or β-galactosidase (β-gal). Efficiency of gene transfer increased with increasing multiplicities of infection and incubation time, with 45% of MKs expressing GFP 72 hours after viral infection. Transduced MKs remained capable of agonist induced GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist responsive GPIIb-IIIa receptors, (2) are capable of expressing transgenes, and (3) may prove useful for investigation of the molecular basis of MK differentiation and GPIIb-IIIa function.

Ex Vivo Cultured Megakaryocytes Express Functional Glycoprotein IIb-IIIa Receptors and Are Capable of Adenovirus-Mediated Transgene Expression

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4084-4092 ◽  
Author(s):  
Nauder Faraday ◽  
Jeffrey J. Rade ◽  
David C. Johns ◽  
Gopal Khetawat ◽  
Stephen J. Noga ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Blood Platelets ◽  
Green Fluorescence Protein ◽  
Hematopoietic Stem ◽  
Development Factor ◽  
Fibrinogen Binding ◽  
Activation Characteristic ◽  
Fluorescence Protein

Abstract Investigation of the molecular basis of megakaryocyte (MK) and platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence of pegylated recombinant human MK growth and development factor (PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as assessed by agonist inducible soluble fibrinogen and PAC1 binding. The specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa receptors were similar to those described for blood platelets. The reversibility and internalization of ligands bound to MK GPIIb-IIIa also shared similarities with those observed in platelets. Cultured MKs were transduced with an adenoviral vector encoding green fluorescence protein (GFP) or β-galactosidase (β-gal). Efficiency of gene transfer increased with increasing multiplicities of infection and incubation time, with 45% of MKs expressing GFP 72 hours after viral infection. Transduced MKs remained capable of agonist induced GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist responsive GPIIb-IIIa receptors, (2) are capable of expressing transgenes, and (3) may prove useful for investigation of the molecular basis of MK differentiation and GPIIb-IIIa function.

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5143-5143
Author(s):  
Liesbeth De Waele ◽  
Kathleen Freson ◽  
Chantal Thys ◽  
Christel Van Geet ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Phosphoglycerate Kinase ◽  
Lentiviral Vectors ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Platelet Disorders

Abstract The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages. Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

scholarly journals In vivo gene transfer into rat bone marrow progenitor cells using rSV40 viral vectors

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2655-2662 ◽  
Author(s):  
Bianling Liu ◽  
Judy Daviau ◽  
Carmen N. Nichols ◽  
David S. Strayer
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Simian Virus 40 ◽  
Hematopoietic Stem ◽  
Entire Study

AbstractHematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by cytokine-induced loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Here, we attempted to circumvent such limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)–derived gene delivery vectors were used because they transduce resting CD34+ cells very efficiently. Rats received SV-(Nef-FLAG), carrying FLAG marker epitope—or a control recombinant SV40 (rSV40)—directly into both femoral marrow cavities. Intracellular transgene expression by peripheral blood (PB) or BM cells was detected by cytofluorimetry. An average of 5.3% PB leukocytes expressed FLAG for the entire study—56 weeks. Transgene expression was sustained in multiple cell lineages, including granulocytes (average, 3.3% of leukocytes, 20.4% of granulocytes), CD3+ T lymphocytes (average, 0.53% of leukocytes, 1% of total T cells), and CD45R+ B lymphocytes, indicating gene transfer to long-lived progenitor cells with multilineage capacity. An average of 15% of femoral marrow cells expressed FLAG up to 16.5 months after transduction. Thus, direct intramarrow administration of rSV40s yields efficient gene transfer to rat BM progenitor cells and may be worthy of further investigation.

Identification of Hepatic Progenitors Having No Hematopoietic Potential in Murine Bone Marrow.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1684-1684
Author(s):  
Yasuhiro Yamada ◽  
Hirosada Miyake ◽  
Eishi Nishimoto ◽  
Hiroaki Mitsuya ◽  
Yuji Yonemura
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Green Fluorescence Protein ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Fluorescence Protein ◽  
Fetal Liver Cells ◽  
Titration Assay

Abstract We recently reported that when Sca-1+ c-Kit− bone marrow cells (BMCs) were co-cultured with fetal liver cells (FLCs) on laminin-coated dishes, alpha-fetoprotein (AFP)-expressing BMCs became completely adherent by day 3 and expressed albumin as assessed with immunochemistry and RNA-PCR (Yamada et al., Exp Hematol. 34: 97, 2006). In the current study, we attempted to further delineate the characteristics of BMCs that differentiate into hepatic-like cells. It was found that AFP-expressing cells were in CD5+ or B220+ lymphoid lineage, mostly Sca-1+CD5+ lineage, expressing AFP. When cKit+Sca-1+ lineage− BMCs (KSLs), which did not express AFP, were cocultured with CD5+ BMCs, both from green fluorescence protein (GFP)-expressing transgenic mice, in the presence of FLCs from ROSA26 mice (X-gal+ FLCs), fractionated cells gave rise to adherent hepatic-like cells, which expressed albumin and cytokeratin 8 (assessed with immunochemistry) and AFP, albumin, transthyretin and dipeptidylpeptidase IV (examined with RNA-PCR). The hepatic-like cells from KSLs and CD5+ BMCs emerged at the frequency of 1 in 50 and 1x103 as assessed with titration assay. However, CD5+ Mac-1− Gr-1− Ter119− BMCs did not differentiate into hepatic-like cells. CD5+ CD45− cells differentiated into hepatic-like cells without fusion at the frequency of 1 in 300 but CD5+ CD45+ cells did not. CD5+ CD45− cells hardly produced hematopoietic colonies as compared with CD5+ CD45+ cells did. In conclusion, we have shown that KSLs and CD5+ CD45− cells exposed to FLCs are capable of generating hepatic-like cells, which expressed albumin as assessed with immunochemistry and RNA-PCR. We should further explore whether CD5+ CD45− cells emerge from hematopoietic stem cells or mesenchymal stem cells.

scholarly journals Application of conditioned medium from mesenchymal stromal cells in the protocol for ex vivo production of megakaryocytes and platelets

2021 ◽  
Vol 66 (4) ◽  
pp. 526-538
Author(s):  
D. Yu. Klyuchnikov ◽  
M. Yu. Yazykova ◽  
A. A. Stepanov ◽  
S. E. Volchkov ◽  
O. V. Tyumina
Keyword(s):  
Cord Blood ◽  
Conditioned Medium ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Blood Platelets ◽  
Venous Blood ◽  
Hematopoietic Stem ◽  
Group 2 ◽  
Group 1

Introduction. Of interest is the use of a conditioned medium from mesenchymal stromal cells in order to increase the expansion of CD34+  hematopoietic stem cells (HSCs).Aim — to analyze the effi cacy of two methods of ex vivo production of human megakaryocytes and platelets from CD34+ cord blood HSC using conditioned media from mesenchymal stromal cells and IMDM. Methods. Two cultivation methods that differ from each other by medium composition were compared. As a control of antigen expression of the donor, venous blood platelets were used. CD34+ HSCs were isolated from mononuclear fraction of cord blood using the immunomagnetic selection technique. The resulting cells were introduced at a concentration of 1 × 104  cells/mL into 24-well plates and cultured at 39 °C and 10 % CO2  for the first 7 days, after which the conditions were changed to 37 °C and 5 % CO2  and cultured for 14 days. In Group 1, up to day 7, the culture was performed using conditioned medium from mesenchymal stromal cell containing TPO (30 ng/mL), SCF (2 ng/mL), IL-6 (7.5 ng/mL), IL-9 (13.5 ng/mL), and in Group 2 a IMDM medium with the same cytokine cocktail was used. The cells were calculated using haemocytometer. CD34, CD41a, CD42b expression was evaluated using fl ow cytometry. Statistic data was processed with using R-language. The differences were evaluated as statistically signifi cant at signifi cance level p < 0.05.Results. Megakaryocyte production was observed starting from day 7 of culture. The expression level using conditioned medium from mesenchymal stromal cells (Group 1) according to CD41a was 5.84 ± 0.33 % versus 10.43 ± 1.08 % using IMDM medium (Group 2). On day 13 the ratio increased up to 42.05 ± 1.71 % in Group 1 and 61.78 ± 1.71 % in Group 2. CD41a+ megakaryocytes of Group 1 expressed the CD42b marker at the level of 96.85 ± 1.06 % versus 88.7 ± 0.56 % in Group 2. With the application of MSC conditioned medium the average number of nucleated cells was signifi cantly higher on the day 11 and it was equal 326.016 ± 1.86 × 104  cells/mL vs 197.26 ± 10.55 × 104  cells/mL in IMDM medium. Proplatelet formation was observed with microscopy staring from the day 12. The ratio of CD41a+ /CD42b+  platelets was 59.5 ± 3.85 % in conditioned medium, 65.9 ± 8.72 % in IMDM, and 96.11 ± 0.89 % in control platelets derived from venous blood.Conclusion. It was demonstrated that the use of MSC conditioned medium leads to an increase in the expansion of nucleated cells, however it decreases the rate of differentiation in megakaryocytes. 

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

A Review of Evaluating Hematopoietic Stem Cells Derived from Umbilical Cord Blood's Expansion and Homing

2020 ◽  
Vol 15 (3) ◽  
pp. 250-262
Author(s):  
Maryam Islami ◽  
Fatemeh Soleimanifar
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Umbilical Cord ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Hematologic Malignancies ◽  
Future Directions ◽  
Hematopoietic Stem ◽  
Efficient Procedure ◽  
Hematopoietic Recovery

Transplantation of hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB) has been taken into account as a therapeutic approach in patients with hematologic malignancies. Unfortunately, there are limitations concerning HSC transplantation (HSCT), including (a) low contents of UCB-HSCs in a single unit of UCB and (b) defects in UCB-HSC homing to their niche. Therefore, delays are observed in hematopoietic and immunologic recovery and homing. Among numerous strategies proposed, ex vivo expansion of UCB-HSCs to enhance UCB-HSC dose without any differentiation into mature cells is known as an efficient procedure that is able to alter clinical treatments through adjusting transplantation-related results and making them available. Accordingly, culture type, cytokine combinations, O2 level, co-culture with mesenchymal stromal cells (MSCs), as well as gene manipulation of UCB-HSCs can have effects on their expansion and growth. Besides, defects in homing can be resolved by exposing UCB-HSCs to compounds aimed at improving homing. Fucosylation of HSCs before expansion, CXCR4-SDF-1 axis partnership and homing gene involvement are among strategies that all depend on efficiency, reasonable costs, and confirmation of clinical trials. In general, the present study reviewed factors improving the expansion and homing of UCB-HSCs aimed at advancing hematopoietic recovery and expansion in clinical applications and future directions.

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