Megakaryocytic particles and microparticles for in vivo hematopoietic cell and gene therapies
11820968 · 2023-11-21
Inventors
Cpc classification
C12N2521/00
CHEMISTRY; METALLURGY
A01N1/0226
HUMAN NECESSITIES
International classification
Abstract
Applications in transfusion medicine requiring platelets, and hematopoietic stem-cell transplantations require either platelets or enhancement of in vivo platelet biogenesis. Gene therapy applications of hematopoietic stem and progenitor cells (HSPCs) require effective and specific modification of HSPCs by DNA, RNA or other biological molecules. Here we disclose methods for the generation, and modification of megakaryocytic microparticles (MkMPs), proplatelets, preplatelets, platelet-like particles and megakaryocyte extracellular vesicles, that can be used in the aforementioned transfusion and transplantation medicine applications and in gene therapy applications involving hematopoietic stem cells. The biological effects of modified or unmodified MkMPs have never been previously disclosed and thus, this invention claims all biological applications of MkMPs in in vivo therapeutic applications to produce various cells and cell parts, modify various target cells or deliver molecules including drugs to HSPCs and related cells.
Claims
1. A method for enhancing platelet biogenesis in a patient in need thereof, comprising administering to the patient isolated CD62P-megakaryocytic microparticles (MkMPs) generated from megakaryocytes, whereby platelet biogenesis in the patient is enhanced.
2. The method of claim 1, wherein the MkMPs are administered to the patient via intravenous infusion.
3. The method of claim 1, wherein the patient suffers thrombocytopenia.
4. The method of claim 1, wherein the patient undergoes chemotherapy.
5. The method of claim 1, wherein the patient has a genetic or idiopathic disorder.
6. The method of claim 1, further comprising enhancing expansion of hematopoietic stem and progenitor cells (HSPCs) in the patient.
7. The method of claim 1, further comprising administering to the patient hematopoietic stem and progenitor cells (HSPCs).
8. The method of claim 7, wherein the HSPCs are administered to the patient via intravenous infusion.
9. The method of claim 1, wherein the MkMPs have a diameter of 100-1000 nm.
10. The method of claim 1, wherein the MkMPs are stored frozen.
11. The method of claim 1, wherein a native RNA, DNA or protein has been removed from the MkMPs.
12. The method of claim 1, wherein the MkMPs are loaded with an exogenous molecule.
13. The method of claim 12, wherein the exogenous molecule is a biological molecule selected from the group consisting of RNAs, DNAs, proteins and lipids.
14. The method of claim 12, wherein the exogenous molecule is a therapeutic drug.
15. The method of claim 12, further comprising loading the MkMPs with the exogenous molecule.
16. The method of claim 12, further comprising unloading a native RNA, DNA or protein from the MkMPs before the MkMPs are loaded with the exogenous molecule.
17. A method for delivery an exogenous molecule into a patient in need thereof, comprising administering to the patient isolated CD62P-megakaryocytic microparticles (MkMPs) generated from megakaryocytes and loaded with an exogenous molecule, and delivering the exogenous molecule to hematopoietic stem and progenitor cells (HSPCs) in the patient, whereby the exogenous molecule is delivered into the patient.
18. The method of claim 17, wherein the MkMPs are administered to the patient via intravenous infusion.
19. The method of claim 17, wherein the MkMPs have a diameter of 100-1000 nm.
20. The method of claim 17, wherein the MkMPs have a diameter of 40-100 nm.
21. The method of claim 17, wherein the exogenous molecule is a biological molecule selected from the group consisting of RNAs, DNAs, proteins and lipids.
22. The method of claim 17, wherein the exogenous molecule is a therapeutic drug.
23. The method of claim 17, further comprising loading the MkMPs with the exogenous molecule.
24. The method of claim 17, further comprising unloading a native RNA, DNA or protein from the MkMPs before the MkMPs are loaded with the exogenous molecule.
25. The method of claim 1, wherein the MkMPs are loaded with an exogenous molecule, the method further comprising delivering the exogenous molecule to hematopoietic stem and progenitor cells (HSPCs) in the patient.
26. The method of claim 17, wherein the patient is in need of enhancing platelet biogenesis.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
(25) While the present disclosure may be susceptible to embodiments in different forms, and herein various embodiments will be described in detail with the understanding that the present description is to be considered an exemplification of the principles of the disclosure and is not intended to be exhaustive or to limit the disclosure to the details of construction and the arrangements of the components set forth in the following description or illustrated in the drawings.
(26) Methods Used for the Disclosure and Enablement of the Invention
(27) Materials and Proteins:
(28) All chemicals were obtained from Sigma-Aldrich or otherwise indicated. Recombinant human interleukin 3 (IL-3), IL-6, IL-9, IL-11, stem cell factor (SCF), granulocyte colony-stimulating factor (rhG-CSF) and thrombopoietin (TPO) were from purchased from PeproTech Inc. Purified human von Willbrand Factor (vWF, Factor VIII free) was from Haematologic Technologies. Human fibrinogen for coverslip coating was from Innovative Research. Alexa Fluor® 647-conjugated fibrinogen for platelet functionality assays was from Life Technologies. Phycoerythrin (PE)-conjugated Annexin V was from BD Bioscience. Human thrombin was from Sigma-Aldrich. Human thrombin was from Sigma-Aldrich. Size standard fluorescent beads (0.22, 0.45, 0.88 and 1.34 μm) and AccuCount fluorescent particles (5.1 μm) were from SpheroTech.
(29) Antibodies:
(30) Fluorescein isothiocyanate (FITC)-conjugated anti-CD41a (GPaIIb), PE-conjugated anti-CD42b (GPIbα), PE-conjugated anti-CD62P, allophycocyanin (APC)-conjugated anti-BrdU (BrdU APC flow kit), APC-conjugated anti-CD34, PE-conjugated CD11b and APC-conjugated anti-CD235a antibodies were from BD Bioscience. Anti-active caspase-3 antibody (ab13847), anti-human β31 tubulin antibody (ab96008), anti-human vWF antibody (ab9378) and anti-serotonin antibody (ab66047) were all from Abcam. The secondary antibodies, Alexa Fluor® 488-conjugated anti-rabbit IgG antibody and anti-goat IgG antibody, were from Life Technologies. FITC-conjugated CD41 antibody for CD41.sup.+-cell enrichment and platelet functionality assays was from Beckman Coulter.
(31) Megakaryocytic Cultures:
(32) Frozen G-SCF mobilized peripheral blood CD34.sup.+ cells were obtained from the Fred Hutchinson Cancer Research Center. CD34.sup.+ cells were cultured using the protocol previously described. Briefly, from day 0 (d0) to d5, cells were cultured in Iscove modified Dulbecco medium (IMDM, GlutaMax™; Life Technologies), pH 7.2, supplemented with 20% BIT9500 (Stemcell Technologies), 100 ng/mL rhTPO, 100 ng/mL rhSCF, 2.5 ng/mL rhIL-3, 10 ng/mL rhIL-6, 10 ng/mL rhIL-11 and 1 μg/mL human low density lipoprotein (hLDL), at 37° C. in fully humidified incubator under 5% CO.sub.2 and 5% O.sub.2. Then from d5 to d7, culture medium was changed to IMDM, pH 7.4, supplemented with 20% BIT9500, 100 ng/mL rhTPO, 100 ng/mL rhSCF, 10 ng/mL rhIL-3, 10 ng/mL rhIL-9, 10 ng/mL rhIL-11 and 1 μg/mL hLDL, and O.sub.2 level was increased to 20%. At d7, CD61.sup.+ cells (Mks) were enriched using anti-CD61 magnetic microbeads (Miltenyi Biotec) and LD magnetic columns (Miltenyi Biotec). After enrichment, Mks (CD41.sup.+ purity >90%) were cultured in IMDM, pH 7.6, supplemented with 20% BIT9500, 100 ng/mL rhTPO, 100 ng/mL rhSCF, 1 μg/mL hLDL and 6.25 mM nicotinamide. From d8 to d12, CD41 and CD62P expression and concentration of microparticles (MPs) in cell culture were measured by flow cytometer (FACSAria II, BD Biosciences) using AccuCount fluorescent particles as internal control.
(33) Shear-Stress Experiments: Exposure of Mk Cells to Shear Flow:
(34) Rectangular flow slides (μ-Slide I.sup.0.6 Luer, ibidi USA) were coated with 50 μg/mL vWF, and ca. 300,000 cultured Mks were seeded into each slide. Mks on slides were cultured overnight (21 hours) before being exposed to shear flow. Medium (IMDM supplemented with 10% BIT9500, 50 ng/mL TPO, 50 ng/mL rhSCF, 0.5 μg/mL hLDL and 6.25 mM nicotinamide) was perfused over Mks on slides by two syringe pumps (Dual NE-4000 pump; New Era Pump Systems) to achieve the desirable shear-stress level. For BrdU incorporation assays, the medium was supplemented with 10 μM BrdU (BD). During shear flow, some Mks were detached from the slide surface and released into the circulating medium. These are considered as non-adherent Mks. Adherent Mks were harvested for analysis using non-enzymatic cell dissociation buffer (Sigma-Aldrich). In some experiments, adherent Mks were fixed with 2% paraformaldehyde directly on slides and processed for immunofluorescence analysis. In some experiments, Mks were treated with caspase inhibitors, 10 μM z-VAD.fmk (Bachem) or 10 μM z-DEVD.fmk (Bachem) starting on d9. Inhibitor-treated Mks were seeded into flow slides at d9 or d11, were exposed to shear flow (2.5 dyn/cm.sup.2 for 0.5 hour) in medium supplemented with the same inhibitor, and were harvested for PPT, PLP and CD41.sup.+ microparticle counting.
(35) DNA Synthesis Assay:
(36) DNA synthesis was assessed using a BrdU APC flow kit (BD Bioscience). After exposure to shear flow for the indicated time, Mks were cultured for additional time period to a total 4 of hours with BrdU in the medium before harvesting for analysis. Cells from static cultures were treated the same way and served as control
(37) Annexin V Assay:
(38) After shear flow application or static control, cells were harvested immediately and stained with FITC-anti-CD41a antibody and PE-Annexin V for flow-cytometric analysis.
(39) Immunofluorescent Staining:
(40) For β1 tubulin staining, cells were fixed and permeabilized using 1% glutaraldehyde and 0.1% Triton® X-100 (Sigma-Aldrich) in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl.sub.2, pH6.9). Then, cells were quenched in 1˜2 mg/mL sodium borohydride before blocking. For other staining, cells were fixed with PFA and permeabilized with Triton X-100. After blocking with BSA (Fisher Scientific) together with normal goat or donkey serum (MP Biomedicals), primary antibodies (active caspase-3, β1 tubulin, vWF or serotonin) or corresponding isotype controls were applied to cells overnight at 4° C., followed by incubation with the secondary antibody conjugated with Alexa Fluor® 488 at room temperature for 1 hour. F-actin and DNA were stained with Alexa Fluor® 568-phalloidin (Life Technologies) and TO-PRO®-3 (Life Technologies), respectively. Fluorescent images were collected via a multiphoton confocal microscope (Zeiss 510 NLO). Mean fluorescent intensity (MFI) of active caspase-3 and the average area for a single Mk were quantified using Velocity® Image Analysis Software (Perkin Elmer).
(41) Isolation of PLPs:
(42) Large cells were excluded from PLP preparations by centrifugation at 150×g for 10 minutes. PLPs were then pelleted by centrifugation at 1000×g for 10 minutes from the PLP-enriched supernatant. After one wash, PLPs were resuspended in Tyrode's buffer and used in platelet-stimulation assays. The number of PLPs and PPTs per slide was measured using a Multisizer™ 3 Coulter Counter (Beckman Coulter).
(43) Platelet-Stimulation Assays: CD62P Exposure and Fibrinogen Binding:
(44) These assays were carried out as described, whereby CD62P expression and fibrinogen binding were measured by flow cytometry.
(45) Preparation of Human Platelet and Platelet-Derived Microparticles (PMPs):
(46) Blood for isolation of human platelets was collected by venipuncture from adult human volunteers after providing written informed consent as approved by the Institutional Review Board at University of Delaware (IRB protocol #190471-3). Blood was collected into a 60-cc syringe containing ACD (trisodium citrate, 65 mM; citric acid, 70 mM; dextrose, 100 mM; pH 4.4) at a ratio of 1:6 parts ACD/blood. Anticoagulated blood was spun by centrifugation at 250×g and the supernatant containing platelet rich plasma (PRP) was then pelleted at 750×g (10 minutes), washed once in HEN buffer (10 mM HEPES, pH 6.5, 1 mM EDTA, 150 mM NaCl) containing 0.05 U/ml apyrase and platelets resuspended in HEPES-Tyrode's buffer (137 mM NaCl, 20 mM HEPES, 5.6 mM glucose, 1 g/l BSA, 1 mM MgCl2, 2.7 mM KCl, 3.3 mM NaH2PO4) at a concentration of 4×10.sup.8 platelets/ml in HEPES-Tyrode's buffer containing 0.05 U/ml apyrase. 1 mM CaCl.sub.2) was added to platelet before activation and platelets were activated by 2 U/mL human thrombin or 10 μM Calcium Ionophore (A23187, Sigma-Aldrich). The platelets were removed by centrifugation at 1000×g for 10 minutes and PMPs were harvested from supernatant washed two times using IMDM medium by ultracentrifugation at 25,000 rpm for 1 hour, 4° C. The concentration of PMPs was measured by flow cytometry using 1.34 μm-diameter microbeads.
(47) ELISA Assay for TPO:
(48) Protein lysates and supernatants from concentrated microparticle suspensions were analyzed using human TPO ELISA (PeproTech) according to manufacturer's protocol. The signal was read at 405 nm on a PerkinElmer Victor 3V multilabel counter.
(49) Isolation and Characterization of MkMPs:
(50) For both static cultures and cultures exposed to shear flow, Mk cells were removed from the culture medium by centrifugation (150×g for 10 minutes). Following that, PLPs were removed by centrifugation at 1000×g for 10 minutes. Particles were then washed twice in IMDM medium and were enriched for MkMPs by ultracentrifugation (25,000 rpm for 1 hour at 4° C.; Beckman Coulter Optima Max Ultracentrifuge). CD41, CD42b and CD62P expression was examined by flow cytometry. Concentrations of MkMPs (and of PMPs and Mks) were measured by flow cytometry using 1.34 μm microbeads (Sphero Tech) as standard. For some experiments, MkMPs in supernatant were incubated with 1 U/mL RNase A/T1 cocktail (Life Technologies) or 10 U/mL RNase ONE™ (Promega) for 1 hour at 37° C. before enrichment.
(51) Human Umbilical Vascular Endothelial Cells (HUVECs), Mesenchymal Stem Cells (MSCs) and Granulocytic Cultures:
(52) Primary HUVECs were obtained from ATCC and cultured according to ATCC recommendation (growth medium from ATCC: vascular cell basal medium supplemented with endothelial cell growth kit-VEGF). Human MSCs were obtained from Lonza and cultured according to Lonza recommendation (growth medium: mesenchymal stem cell basal medium supplemented with MSCGM™ SingleQuots™). Human granulocytes were differentiated from human CD34.sup.+ cells as previously described. Human long-term medium (HLTM) was prepared by supplementing McCoy's 5A medium with 12.5% heat-inactivated (57° C. for 30 minutes) fetal bovine serum (Hyclone), 12.5% heat-inactivated horse serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 1% minimal essential medium (MEM) essential amino acid solution (Life Technologies), 1% MEM nonessential amino acid solution (Life Technologies), 1% MEM vitamin solution (Life Technologies), 100 mM monothioglycerol, 10 mM HEPES, and 50 mg/mL gentamycin sulfate. CD34.sup.+ cells were cultured in HLTM supplemented with 50 ng/mL rhSCF, 10 ng/mL rhIL-3, rhIL-6 and rhG-CSF (supplement fresh rhG-CSF every 2 days due to degradation) in fully humidified incubator under 5% CO.sub.2 and 5% O.sub.2. At d7 of cell culture, CD15.sup.+ cells were enriched using MS column and CD15 microbeads (Miltenyi Biotec).
(53) Mk Ploidy Analysis:
(54) Cells from MkMP coculture were stained with FITC anti-CD41 antibody before fixation by 0.5% paraformaldehyde (Electron Microscopy Sciences) and permeabilization by 70% methanol/H.sub.2O. After RNA was degraded by RNase A (Life Technologies), DNA was stained with 100 g/mL propidium iodide. Analyses of CD41 expression level, cell ploidy and numbers were performed on flow cytometry using AccuCount fluorescent particles as internal control.
(55) MkMP Binding and Uptake Analysis:
(56) MkMPs were stained with 20 μM CFDA-SE (Life Technologies) for 20 minutes at 37° C. and washed three times in IMDM medium. Then MkMPs were cocultured with HPCs from d3 Mk culture at concentration of 30 MkMPs/cell for indicated time before analysis. For the first hour, the coculture medium was 50 μL IMDM and after that coculture was diluted in IMDM supplemented with 5% BIT9500, 50 ng/mL rhSCF and 1% pen strep (Life Technologies). Flow cytometry was used to measure binding of MkMPs to cells. In some experiments, after 3 hours, images of coculture were collected via confocal microscope (Zeiss 5 μLIVE DUO Highspeed/Spectral Confocal, Bioimaging Center, Delaware Biotechnology Institute).
(57) Transmission Electron Microscopy (TEM):
(58) Cells from coculture were fixed in 2% glutaraldehyde and 2% paraformaldehyde in 0.2 cacodylate buffer overnight at 4° C., washed, postfixed within 2% osmium tetroxide for 1 hour at room temperature, followed by 4 washes in H.sub.2O. The samples were then stained en bloc overnight at 4° C. with 1% uranyl acetate. After dehydrated in a series of ascending acetone solutions, samples were infiltrated within n-BGE and then Quetol-NSA resin. Samples were then embed in labeled BEEM capsules and polymerized at 60° C. for 24-48 hours. Ultrathin sections were prepared using a Reichert Jung Ultracut E ultramicrotome, and were collected onto 200 mesh formvar/carbon coated copper grids. Grids were stained with 2% methanolic uranyl acetate and Reynolds' lead citrate. Transmission Electron Microscopy was performed on Zeiss Libra 120 Transmission Electron Microscope and images were acquired using a Gatan Ultrascan 1000 CCD.
(59) Scanning Electron Microscopy (SEM):
(60) The d3 HPCs from Mk culture were incubated with MkMPs (10 MPs/cell) in 100 μL medium for 2 or 4 hours. Then the coculture was let spread on circle coverslip coated with 1 μg/mL human fibronectin for another hour. 2% EM grade glutaraldehyde/IMDM medium was added to coverslips to fix the cells for at least 1 hour at room temperature or overnight at 4° C. Then the samples were washed with PBS and postfixed for 1.5 hours in 1% OsO.sub.4 in H.sub.2O at room temperature. After rinsed with H.sub.2O, the samples were dehydrated in a series of ascending ethanol concentrations for 10 minutes in each solution. After critical-point drying by Autosamdri-815B Critical Point Dryer (Tousimis), the samples were sputter-coated with gold using a Bench Top Turbo III Sputter Coater (Denton Vacuum). The electron images were collected via Hitachi S4700 Field-Emission Scanning Electron Microscope (Hitachi) at a working distance of 8.5-10.5 mm and voltage of 3.0 kV.
(61) Statistical Analysis:
(62) Paired Student t test of all data was performed by Minitab 16 (Minitab). Statistical significance was defined as P<0.05.
Example 1. Shear Flow Promotes DNA Synthesis and Accelerates the Polyploidization of Mks in a Largely Dose and Maturation-Stage Dependent Way
(63) Mk cells engage endomitosis as they mature and become polyploid. We hypothesized that biomechanical forces, such as physiological shear forces, would impact DNA synthesis. To investigate this hypothesis, Mk cells from d7 of culture were seeded onto vWF-coated slides and cultured overnight before exposure to shear flow using perfusion with medium containing 10 μM BrdU. We employed a validated perfusion system (
(64) To investigate if shear flow differentially affects DNA synthesis at different differentiation stages, Mks at d8, d10 and d12 were exposed to 2.5 dyn/cm.sup.2 for 30 minutes. Our data (
(65) We also examined the impact of biomechanical forces on non-adherent cells. In contrast to adherent Mk cells, DNA synthesis of non-adherent Mk cells in these experiments was not affected compared to static conditions (
(66) Could the effect of shear stress on DNA synthesis be due to differential retention of adherent Mk cells because adherent Mks cells were more active in synthesizing DNA? Our data suggest that this is not the case. Indeed, the % CD41.sup.+ cells and ploidy distribution among adherent Mks under various (stress level and duration) flow conditions and static conditions were similar (
(67) We also found that Mk cells respond to higher shear stresses (up t0 400 dyn/cm.sup.2, but more likely up to 100 dyn/cm.sup.2) such as those encountered in the lung vasculature and systemic blood circulation (see Supplemental material of ref. [9]). Mk cells are trapped in the lung vasculature where they experience higher shear and normal stresses than in the bone marrow. Mk cells are also exposed to variable (in magnitude and frequency) shear and normal stresses, from both laminar and turbulent flows (this laminar and turbulent shear and other stresses) in the bone marrow and in systemic blood circulation due to the pulsatile blood flow, different blood vessel diameters and due to squeezing through blood-vessel endothelial cells (see Refs [1, 9] and Supplemental material of ref. [9]). Shear stresses can range from 0.1 to 100 dyn/cm2. Frequency of stress application can range from a few seconds (10, 20, 30, 60, 120 seconds to a few minutes, 1-10 minutes) depending on location in the blood and lung vasculatures and the trapping of Mk cells between other cells. All such biomechanical stresses of variable magnitude and frequency can stimulate Mk cells and can lead to increased DNA synthesis and the formation of various particles derived from Mk cells as described in the examples below.
(68) Shear flow is a flow of fluid in a channel that creates a shear stress on the walls of the channel or on the surfaces of objects (such as cells or particles) in the flow channel. Shear stress, here due to fluid flow, is a mechanical stress that arises in a flow field in a channel or around an object (such as a cell or particle) in the flow field due to the changing fluid velocity in the channel in any cross section of the channel or for the case of flow around an object due to the changing velocity in the area of the flow field near the object. The shear stress is tangent to the fluid element surface. The shear stress is highest on the wall of the channel where the velocity changes the fastest, as in this case the cells are grown. A shear stress is also the highest near the surface of an object in a flow field. In a flow, the normal stress is perpendicular to the surface of the fluid element in a flow field. A laminar shear stress arises in a laminar fluid flow, which is the fluid flow where the fluid flows in parallel thin layers, with no disruption between the thin layers. Turbulent stresses arise in the complex fluid patterns of turbulent fluid flow, in which there are eddies created in the flow that create a chaotic pattern of fluid motion and where, in contrast to laminar flow, the fluid does not flow in orderly parallel thin layers.
Example 2. Shear Stress Promotes Phosphatidylserine (PS) Surface Exposure on Maturing Mk Cells
(69) As an early mark of apoptosis, previous studies in our lab and other labs have shown that PS becomes exposed on the extracellular side of the Mk membrane when HPSCs (both human and murine) were differentiated into Mks. In this study, to differentiate human CD34.sup.+ cells into Mks, we used a new protocol that gives rise to functional PLPs in vitro. Using flow cytometry and microscopic analyses, we confirmed that PS is also exposed on the surface of maturing (>d8) Mks generated by this protocol (data not shown). To investigate if shear promotes PS externalization, fluid flow at 1 dyn/cm.sup.2 was applied to d8 and d10 Mks for 2 hours. Cells were harvested immediately after the shear-flow application for analysis. Shear resulted in a significantly increased fraction (by ca. 160% and 260% at d8 and d10, respectively) of adherent Mks that are Annexin V.sup.+, but not so for non-adherent Mks (
Example 3. Caspase-3 Activation in Maturing Mk Cells is Accelerated by Exposure to Shear Flow
(70) It has been shown that activation of caspase-3 and 9 is required for PPT formation. Here we wanted to investigate if shear stress affects caspase-3 activation in Mks. We chose caspase-3 as a marker of late apoptosis to complement our Annexin V studies above, which pertain to early apoptotic events. First, we confirmed that caspase-3 in indeed activated during in vitro Mk maturation with our culture protocol. Caspase-3 was activated at d10 and d12 when Mks projected PPTs under our culture protocol; active caspase-3 accumulated largely around the nucleus, but PPTs did not stain for active caspase-3 (
Example 4. Shear Stress Enhances the Generation of Functional Platelet-Like Particles (PLPs) as Well PLP Activity
(71) Here we aimed to investigate and quantify the effect of shear stress on the generation of Mk fragments with platelet-like properties at d12 when Mks had extensive PPTs (
(72) Next, we examined the impact of shear flow on the functionality of the generated PLPs. Is it possible that the fast generation of PLPs under shear flow results in lesser quality of PLPs, or perhaps the opposite? To do so, we employed two platelet-function assays, CD62P exposure and fibrinogen binding assays, both using the physiological activator: human thrombin. For PLPs generated from Mks under 1 dyn/cm.sup.2 for 2 hours, the fraction of PLPs expressing CD62P increased by almost 3-fold (from 10% to 29%) upon thrombin activation, while for PLPs generated from Mk cells under static conditions this fraction increased by 1.8-fold (from 9% to 16%;
(73) We also quantified the Mk-fragmentation outcomes aiming to illuminate and support the data of
Example 5. Shear Stress Dramatically Enhances the Generation of Mk-Derived Microparticles (MkMPs)
(74) When we examined the size distribution of cell fragments released from Mks under both static and shear-flow conditions, in addition to PLPs (d=1-3 μm) and PPTs (d=3-10 μm), we found a distinct population of very small particles (
(75) Cultures of Mk cells post shear exposure contained a dramatically larger number of these MkMPs compared to those from Mks grown under static conditions (
(76) Flaumenhaft et al. have shown that the CD41.sup.+ MPs in human plasma are mainly derived from Mks rather than activated platelets [5]. However, no mechanism for generating MkMPs was previously known, and no function for MkMPs was known either until this present set of investigations and data. Our data support the thinking that when mature Mks enter BM sinusoids and are exposed to shear circulatory forces, numerous MkMPs are likely formed. While PMP generation from platelets on immobilized von Willebrand-factor (vWF) coated surfaces under high shear was previously shown, it was reported that vWF was necessary for the generation of PMPs under shear flow. These findings pertain to the generation of MkMPs (which are different from the PMPs) under shear flow and without the need for vWF involvement. While the cellular mechanisms leading to membrane vesiculation and MP release remain an active research field, studies from PMP biogenesis suggest that PS externalization and caspase-3 activation play an important role in MP generation. In our study, we found that caspase-3 activation and PS externalization were enhanced by shear stress, thus suggesting that shear-stress enhanced MkMP generation may be mediated by PS externalization and caspase-3 activation. The latter is supported by the data from the caspase-3 inhibition assays.
Example 6. Novel Biological Activity of MkMPs: Promoting Mk Differentiation of HSPCs
(77) A physiological function for MkMPs has not been yet previously reported. This is the first study to identify the role and potential use of MkMPs. We hypothesized that a role of MkMPs might be to accelerate hematopoietic-progenitor differentiation into Mks. In early experiments, we found that MkMPs cocultured with HPCs from d5 of Mk culture from CD34.sup.+ cells promoted HPC survival and Mk differentiation under serum- and TPO-free conditions. We thus examined in more detail this effect using MPs generated from d12 Mks. We will refer to these MPs as MkMPs although they may contain a small fraction (ca. 16%) of CD62P.sup.+ MPs. MkMPs were cocultured with CD34.sup.+ cells in a medium without added TPO but with 50 ng/mL rhSCF (for enhancing cell survival), and the outcomes were examined after 8 days of culture. In more detail, 30,000 CD34.sup.+ cells (or cultured HPCs from d3 culture of CD34.sup.+ cells with or without TPO) were incubated with 10 MkMPs or PMPs per CD34.sup.+ cell or HPC in 50 μL IMDM medium for 1 hour at 37° C. to enhance the contact between MPs and cells. After that, the cells with the MPs were diluted in 300 μL IMDM medium supplemented with 5% BIT9500 and 50 ng/mL rhSCF and cultured at 37° C. and 20% O.sub.2. For some coculture, Lin.sup.+ cells (CD2.sup.+, CD3.sup.+, CD11 b.sup.+, CD14.sup.+, CD15+, CD16.sup.+, CD19.sup.+, CD56.sup.+, CD123.sup.+, or CD235a.sup.+) from CD34.sup.+ cells before coculture with MkMPs. For some coculture of MkMPs and d3 HPCs, cells were harvested after 5 hours of incubation and then processed for TEM imaging. For some cocultures, MkMPs were labeled with fluorescent dye CFDA-SE (Sigma-Aldrich) first and incubated with d3 HPCs for various times before analysis by flow cytometry. For other coculture, cells were harvested on d8 for CD41 and ploidy flow-cytometry analysis. At d9, cells in coculture were examined using multiphoton confocal microscope (Zeiss 510 NLO), and DIC (Differential Interference Contrast) images were collected. At d10, cells from coculture were seeded onto human fibrinogen-coated coverslips and cultured overnight for staining for β1 tubulin (TUBB1), vWF and serotonin (5-HT) at d11. Cells from vehicle control were fixed first and cytospun onto coverslip using Shandon Cytospin 4 (Thermo Scientific) before immunofluorescent staining. At d11, some cells were harvested for TEM imaging.
(78) In the vehicle control culture, barely any CD34.sup.+ cells could differentiate into Mks by d8 (
(79) In order to further characterize the Mks generated from CD34.sup.+ cells cocultured with MkMPs, the coculture was prolonged to d11. At d9, we found that some Mks started to form proplatelets (
(80) Since d12 MkMPs contained ca.16% CD62P.sup.+ MPs with PMP characteristics, we examined if PMPs generated from activated human platelets by thrombin or the calcium ionophore A23187 could have an effect similar to that of MkMPs. Compared to vehicle control, coculture of d3 HPCs (from cultures with or without TPO) with either type of PMPs did not affect the Mk differentiation of HPCs compared to control (
(81) Although the protocol for generating MkMPs employs rigorous centrifugal enrichment and triple washing in IMDM medium, we wanted to verify that the impact of MkMPs in promoting the Mk differentiation of HSPCs was not due to TPO attached to MkMPs. To this effect, we used a TPO ELISA assay to measure the amount of TPO carried by MkMPs and PMPs. We found that the total TPO carried into the cocultures by the MPs would result in 5.2, 14, 27 pg/mL TPO in HSPC cocultures with PMPs (A23187), PMPs (thrombin) and MkMPs, respectively. This assumes that all TPO becomes available to all HSPCs, which is not true as we found that many MkMPs remain in culture for many days without being attached to cells. No study has tested the effect of TPO at such low concentrations. The lowest TPO level examined is 100 pg/ml, which has only a small impact on Mk differentiation compared to saturation TPO levels. In support of the argument that the impact of MkMPs does not derive from the small amount of TPO carried into the coculture, we note that the TPO carried by the thrombin-generated PMPs (which had no Mk-differentiation impact on HPCs) was about half that carried by MkMPs.
(82) This is the first study ever to show that true MkMPs have a biological role in inducing megakaryocytic differentiation of HSPCs in the absence of TPO and to do so in a physiological significant way leading to the formation of biologically active proplatelets as shown in
Example 7: MkMPs are Produced Largely by Mature Mk Cells
(83) We have shown above in Example 6 that Mks can shed CD41+CD62P.sup.− MPs (i.e., MkMPs), and that exposure to fluid shear stress could enhance this process by more than 20 fold in terms of numbers of MkMPs produced. In order to investigate MkMP generation in details under static conditions, CD34.sup.+ HSCs were induced to differentiate into Mks as previously described. At d7 of cell culture, Mks were enriched (CD41.sup.+: >95%) and seeded in fresh medium at concentration of 200,000 cells/mL. Concentration and CD62P expression of CD41.sup.+ MPs in Mk cell culture were measured by flow cytometry from d8 to d12. The data show that more than 85% of CD41.sup.+ MPs in cell culture from d8 to d12 were CD62P.sup.−, indicating that most of CD41.sup.+ MPs were derived from Mks rather than platelet-like particles (PLPs) (
Example 8: Characterization of MkMPs Produced from Mature Mk Cells
(84) We used MkMPs from d12 cell culture in the following studies. Through successive centrifugation, MkMPs were isolated from cell culture and processed for flow cytometric and electron microscopic analyses to obtain the size distribution of MkMPs. The flow cytometry data demonstrate that most of MkMPs were smaller than microbeads with diameter of 0.88 μm (
Example 9: MkMPs Target with Even Higher Effectiveness the Most Primitive Hematopoietic Stem Cells, Namely the CD34+ Lin.SUP.− .Cells. Significantly, MkMPs Also Promote the Expansion of the CD34.SUP.+ HSPCs
(85) In example 6, we showed that MkMPs could induce and enhance differentiation of CD34.sup.+ HSPCs and partially differentiated HPCs from d3 and d5 Mk culture to Mks that were functional to project PPT and synthesize both α- and dense-granules without additional exogenous TPO stimulation. Here, we wanted to show that MkMP cells target also the least differentiated of the HSPC CD34.sup.+ cells, the Lineage negative (Lin.sup.−) cell by removing the committed Lineage positive (Lin.sup.+) cells namely the CD2.sup.+, CD3.sup.+, CD11b.sup.+, CD14.sup.+, CD15.sup.+, CD16.sup.+, CD19.sup.+, CD56.sup.+, CD123.sup.+, or CD235a.sup.+ cells. This was achieved using the lineage cell depletion kit from Miltenyi Biotec. A total of 60,000 CD34.sup.+ Lin.sup.− cells were incubated with 10 MkMPs/cell in 50 μL IMDM medium for 1 hour at 37° C. to enhance the contact between MkMPs and target cells. After that, coculture of MkMPs with CD34.sup.+ Lin.sup.− cells were diluted in 600 μL IMDM medium supplemented with 5% BIT9500 and 50 ng/mL rhSCF, and cultured at 37° C. and 20% 02. All cocultures were maintained for 8 days before harvest for ploidy assay and analysis by flow cytometry. CD34.sup.+ Lin.sup.− cells at d0 and cells from coculture at d3, d5 and d8 were stained with CD41, CD34, CD11 b and CD235a antibodies and analyzed by flow cytometry. Flow cytometry analysis shows that MkMP coculture had a large amount of Mks with 2N, 4N and >=8N ploidy classes while very few Mks were found in the vehicle control culture (
(86) In order to find out what are the CD41.sup.− cells are in the MkMP coculture, cells from the cocultures and control cultures were stained with anti-CD34, CD41, CD11 b and CD235a antibodies to identify HSPCs, Mks, granulocytes and erythrocytes, respectively, and analyzed by flow cytometry. The results show that very few cells differentiated into granulocytes or erythrocytes in either the vehicle control culture or the MkMP coculture, indicating that MkMPs could not induce HSCs differentiation to these two lineages. This shows that the effect of MkMPs on HSPCs is specific to the Mk lineage differentiation and supports the claims related to outcome specificity for in vivo applications, i.e., that MkMPs promote ONLY the Mk differentiation of HSPCs.
(87) In the MkMP coculture, the percentage of CD41.sup.+ cells increased from 0% at d0 to ˜47% at d5 and plateaued after d5, indicating that 5 days are sufficient for HSCs to commit to Mk lineage induced by MkMPs. From the ploidy analysis, the percentage of CD41.sup.+ cells in coculture at d8 was about 19% which is lower than the ˜48% obtained from the surface marker staining analysis. This could be due to the assay methodology since we always obtained lower CD41 percentages from ploidy analysis than surface marker staining. This could be also contributed partially by the possibility that CD41.sup.− cells with CD41.sup.+ MkMPs attached were detected by flow cytometry as CD41.sup.+ cells in surface-marker staining analysis but not in ploidy assay. Compared to the vehicle control culture, the coculture also had a higher percentage of CD34.sup.+ cells at d8 (47% vs. 28%) and based on the total cell number obtained from ploidy assay, there were more CD34.sup.+ cells in coculture than control culture (108 k vs. 27 k,
Example 10. Target Specificity: MkMPs could not Trans-Differentiate Human Granulocytes, MSCs and HUVECs into Mk Cells
(88) Previous studies have demonstrated that certain types of cells can trans-differentiate into other unrelated types of cells It is possible that cell-derived microparticles mediate trans-differentiation. For example, microparticles derived from lung endothelial cells induced trans-differentiation of bone marrow cells into endothelial cells. To investigate if MkMPs could trans-differentiate other cell types into Mks, we tested human granulocytes, MSCs and HUVECs, all of which are encountered by MkMPs in the bone marrow environment or in circulation.
(89) A total of 60,000 HUVECs (human umbilical cord vascular endothelial cells; passage 3-5, obtained from ATCC), MSCs (mesenchymal stem cells; passage 2-4, donation from Prof. Xinqiao Jia, Univ of Delaware) or enriched CD15.sup.+ granulocytes were incubated with 10 MkMPs/cell in 50 μL IMDM medium for 1 hour at 37° C. to enhance the contact between MPs and cells. After that, coculture of MkMPs with MSCs and granulocytes were diluted in 600 μL IMDM medium supplemented with 5% BIT9500 and 50 ng/mL rhSCF, and cultured at 37° C. and 20% 02 and coculture of MkMPs with HUVECs were diluted in 600 μL growth medium without any endothelial cell growth factors. All cocultures were maintained for 8 days before harvest for ploidy assay and analysis by flow cytometry. Flow cytometry analysis (
Example 11. MkMPs Promote Mk Differentiation Through Transfer of the RNA Carried by the MkMPs
(90) Several studies have reported that signaling molecules carried by MPs, including ESC-derived MPs, MSC-derived MP and PMPs, are RNA (mRNA and/or miRNA) and MPs exert their biological function through RNA transfer to target cells. To investigate if MkMPs induce Mk differentiation of HSPCs through RNAs, MkMPs were treated with RNase to degrade if possible the RNA carried by these MkMPs and cocultured with HSPCs, similar to what previous studies have reported. Two different commercial RNases, the RNase A/T1 cocktail from Ambion and the RNase ONE™ from Promega, were used in this study and ploidy analysis was used to examine the effect of RNase treatment. As expected, we found that MkMPs without RNase treatment induced Mk differentiation of HSCs and increased the total cell number while no differentiation was observed in vehicle control culture (
Example 12. MkMPs Interact with Target Cells Through Endocytosis and Membrane Fusion
(91) Next, we examined detailed mechanisms through which MkMPs exerted their impact on HSPCs. Although it has been proposed that MP may interact with or be taken up by target cells through direct fusion and endocytosis, there is no information disclosed as to how MkMPs interact and may transfer molecules they contain to target cells. To investigate the mechanisms by which MkMPs interact with target cells and notably HSPCs, MkMPs were stained with cell cytoplasmic tracker dye CFDA-SE and then cocultured with hematopoietic progenitor cells (HPCs) from d3 of Mk culture. After cultured for certain time as indicated below, cells were processed for flow cytometric and microscopic analyses.
(92) The coculture was firstly analyzed by flow cytometry to examine the kinetics of MkMP binding to cells. At each indicated time point, some cells were harvested from coculture with CFDA-SE stained MkMPs for measurement of mean fluorescence intensity (MFI) of CFDA-SE. The results show that CFDA-SE MFI of cells increased dramatically within one hour of coculture and reached the maximum level at one hour (
(93) Since flow cytometry cannot differentiate MP binding to cells and uptake of MkMPs by cells, coculture of MkMPs and HPCs was examined under confocal microscopy to directly visualize the interaction between live cells and MkMPs. After 3-5 hours of coculture, we observed that most HPCs contained CFDA-SE dye of variable intensity (
(94) In addition to cell internalizing intact MkMPs, we also observed that a gradient of CFDA-SE dye distributed inside some cells and this gradient started from one MkMP on the cell membrane as indicated by the highly concentrated dye (
(95) SEM micrographs of cells from coculture (3-5 hours) show that MkMPs interacted with through a membrane fusion process (
(96) In order to capture the internal structures of the MkMP-cell fusion, cells from coculture were processed for TEM analysis. We captured MkMPs bound to cell surface in TEM micrographs (Data not shown). However, we did not successfully capture MkMP-cell fusion events at all four stages identified in SEM micrographs. A possible reason could be that since TEM examines one ultrathin slice of cell samples, the chance that the ultrathin slice contains one MkMP is low. Nevertheless, two MkMPs fused with cells were found in TEM micrographs (
(97) This is the first disclosure ever as to of the mechanism by which MkMPs interact with and target HSPCs and supports our claims for the use of unmodified and modified MkMPs.
Example 13. Generation of MkMPs (Termed CMPs) from the Human Megakaryocytic Cell Line CHRF and Demonstration that CMPs can Also Induce and Promote Mk Differentiation of HSPCs
(98) CMPs were generated from d3 phorbol 12-myristate 13-acetate (PMA)-induced CHRF cells. 40000 HSCs were coculture with or without CMPs at the concentration of 50 MPs/cell for 1 hr at 37° C. in 50 mL IMDM in the medium to increase CMP-cell contact. Cells with or without CMPs were then diluted into 600 mL medium without thrombopoietin (IMDM, 5% BIT9500, 50 ng/mL rhSCF) at 37° C. and 20% O.sub.2 for 8 days. Cell were harvested on d8 for CD41 and DNA staining and analyzed of CD41 expression and ploidy by flow-cytometry (FACSAria II, BD bioscience). Collected CMPs were cocultured with HSCs at the concentration of 50 MPs/cell for 8 days. From the analysis of ploidy flow-cytometry,
Example 14. RNase Treatment is Effective in Eliminating (“Unloading”) the Native RNA in Megakaryocytic Microparticles so that they can be Loaded with Desirable Molecules for Delivery to Target HSPCs
(99) We used CMPs as a model for primate MkMPs. In example 11, we have shown that RNase treatment can partially abrogate the impact of MkMPs on HSPCS. Here we optimized the process of RNase treatment to remove the RNA content of CMPs. HSPCs were coculture with CMPs, or RNase-treated CMPs, or without CMPs (Control) for 8 days. In detail, CMPs were collected as mentioned previously, and were treated with 1 U/mL RNase A/T1 (Ambion) or 10 U/mL RNase ONE (Promega) under the condition of 37° C. for 1 hr. After that, 10 U/mL RNase inhibitor, SUPERase-In (Ambion) were added to prevent further reaction from RNase. CMPs were then washed with IMDM and collected by ultracentrifugation at 25000 rpm, 4° C. 60000 of HSCs were cocultured with CMPs, or RNase-treated CMPs, or without CMPs at the concentration of 50 MPs/cell for 8 days in the IMDM medium supplemented with 5% BIT9500, 50 ng/mL rhSCF, but without thrombopoietin. Cell were harvest at d8 for CD41 and DNA staining. Analysis of cell ploidy and CD41 expression and Mk cell number were performed by flow-cytometry (FACSAria II, BD bioscience). HSPCs cocultured with CMPs without RNase treatment became Mks with polyploidy and CD41 positive and the number of Mks with CMPs coculture is higher than Mks of vehicle control. However, here, the number of Mks of both RNase-treated CMPs cocultures decrease, compared to the Mks in CMP coculture (
Example 15. Loading of pmaxGFP DNA into CMP or MkMP by Electroporation
(100) To load exogenous material into MP, we choose plasmid DNA as model molecule for loading into MkMPs. There is no prior art on the loading of any microparticles (MkMPs or any other MPs) with exogenous molecules, and thus the following enabling data support our claims for the modification and loading of MkMPs and all MPs with exogenous molecules like DNA, RNA, proteins, non-protein morphogens and drugs. Such loading process could not be anticipated by someone skilled in the art since MPs are very different entities from cells.
(101) We used a commercially available DNA plasmid (PmaxGFP DNA; Lonza), which we labeled with red fluorescent dye Cy5 using the Label IT® Nucleic Acid Labeling Kit (Mirus) based on the manufacturer's protocol. 2 μg of Cy5-pmaxGFP were electroporated into 10.sup.6 CMPs or MkMPs by using AMAXA Nucleofector™ II Device (Lonza) with program T03 or U08, respectively. The procedure of electroporation was followed by manufactural protocol. CMPs or MkMPs were then incubated at 37° C. for 30 min. After 3 times of wash of CMPs or MkMPs with IMDM medium by ultracentrifugation at 25000 rpm, 4° C. for 1 hr each, the loading efficiency of Cy5-pmaxGFP into CMPs or MkMPs were analyzed with flow-cytometry FACSAria II based on Cy5 fluorescence. Electroporation of fluorescent-labeled plasmid DNA (Cy5-pmaxGFP) into MkMPs and CMPs have been performed on AMAXA Nucleofector™ II Device using the specific program U08 and T03, respectively. Base on Cy5 signal, flow-cytometry analysis shows that 95.9% of MkMPs and 97.1% of CMPs were Cy5 positive (
(102) Discussion of Our Disclosed Data for the Support of Our Claims
(103) We show for the first time that shear stress dramatically increased MkMP generation by 30-40 fold. Flaumenhaft et al. demonstrated that the CD41.sup.+ MPs in human plasma are mainly derived from Mks rather than activated platelets [5]. When mature Mks enter BM sinusoids and are exposed to shear circulatory forces, numerous MkMPs are likely formed. PMP generation from platelets on immobilized vWF surfaces is also promoted by high shear. While the cellular mechanisms leading to membrane vesiculation and MP release remain an active research field, studies from PMP biogenesis suggest that PS externalization and caspase-3 activation play an important role in MP generation. In our study, we found that caspase-3 activation and PS externalization were enhanced by shear stress, thus suggesting that shear-stress enhanced MkMP generation may be mediated by PS externalization and caspase-3 activation. The latter is supported by the data from the caspase-3 inhibition assays.
(104) The physiological function for MkMPs was also investigated. We demonstrated that MkMPs promote the survival and Mk differentiation of HSPCs in the absence of added TPO. Thus, one possible role for MkMPs in circulation may be to promote differentiation of circulatory HSPCs or perhaps re-enter the hematopoietic BM compartment aiming to target HSPCs for accelerated megakaryopoiesis under stress. Biological roles have been reported previously for other MPs, but never before for MkMPs. For example, MPs generated during macrophage differentiation of THP-1 cells induced differentiation of resting THP-1 cells into macrophages through miRNA-223 transfer [2].
(105) MPs may serve in several different roles in biological processes since bioactive molecules carried by MPs are concentrated and can travel long distances with protection from degradation. Here, we demonstrated that MPs generated by mature Mks induce Mk differentiation of very primitive HSPCs (CD34.sup.+Lineage.sup.− cells). In addition, we also investigated if MkMPs could transdifferentiate other types of cells, including MSCs, HUVECs and granulocytes and found out they could not. All of these three types of cells are likely to be in contact with MkMPs in vivo.
(106) We also demonstrated mechanisms through which MkMPs exert their biological effect. Two main questions were examined here: how do MkMPs interact with target cells, and what might be the signaling molecules carried by MkMPs. Three different mechanisms have been reported in the literature to explain how cell-derived MPs and target cells interact. The interaction always starts from MP binding to cells, which requires recognition between receptors and ligands on the membrane surface of MPs and cells. This ligand-receptor recognition is the major reason for the target specificity of MPs. MP binding could be unstable, leading to dissociation of MPs from cell surface, or stable, ending in uptake of MPs by cells. The signaling from temporary or persisting binding of MPs could be enough to regulate cell fate. For example, transfer of CCL5 from PMPs to activated endothelial cells only happens during transient interaction rather than firm attachment between PMPs and endothelial cells under flow condition [6]. It is possible that this temporary binding of MkMPs may have an effect on target HSCs. In addition, we have shown that some MkMPs were taken up by cells through direct fusion and/or endocytosis. After we treated MkMPs with two different RNases to digest the RNA carried by MkMPs, the numbers of Mks with different ploidy levels decreased by half in the coculture with treated MkMPs compared to MkMPs without treatment. This result demonstrates that horizontal transfer of RNA is crucial for the observed biological effect of MkMPs. Transfer of RNA requires uptake of MkMPs by cells following stable binding and thus the RNase treatment results provide additional evidence that MkMPs are taken up by cells. Through confocal microscopy, SEM and TEM analyses of MkMP coculture, we demonstrated that both direct fusion and endocytosis were involved in uptake of MkMPs. Moreover, for the first time, using SEM analysis we dissected the MP fusion process as proceeding through 4 distinct stages. The lamellipodia-like structures during MP fusion were observed and reported for the first time. These results contribute to our limited understanding of MP uptake by cells, which is important regarding the delivery of biological molecules (such as but not limited to RNA, DNA, proteins, lipids) as well other organic molecules and drugs by modified MkMPs for delivery to stem cells. A variety of cellular contents may be delivered to target cells including RNA, DNA, proteins, lipids, phospholipids, non-protein morphogens, non-biological materials, organic molecules, non-organic molecules, synthetic drugs or natural drugs.
(107) It has been reported that MPs have several biological functions and play an essential role in various physiological and pathophysiological processes. Different MPs may have a role in blood coagulation, inflammation, angiogenesis, tumorigenesis, cell differentiation and maturation. Ratajczak et al. showed that MPs from embryonic stem cells (ESCs) when cocultured with hematopoietic progenitor cells (HPCs) result in upregulated expression of early marker of pluripotent (Oct-4, Nanog and Rex-1) and early hematopoietic stem cells (Scl, HoxB4 and GATA 2) markers [7]. Their data suggest that the effect is mediated by RNAs and proteins in the MPs. In another example, MPs from stimulated or apoptotic T lymphocytes harbored sonic hedgehog (Hh) morphogens and were able to induce K562 cells (a cancer cell line) differentiation towards the Mk lineage and promote Mk differentiation of CD34.sup.+ HSPCs when cultured in the presence of thrombopoietin [8]. Hh was necessary for these effects. Our invention is very distinct and could not have been anticipated by these findings for several reasons explained in detail below. First, we employ MkMPs and not T-cell derived MPs. Second, our MkMPs do not require Hh morphogens for the effects on HSPCs. Thirdly, this report requires that CD34.sup.+ cells are cultured in the presence of TPO, while our invention does not. Fourth, in this report, they show that Hh containing T-cell MPs promote Mk differentiation but not the production of proplatelets or platelets or platelet-like particles.
(108) Mk cells are great sources for MPs since they have much larger cell volume and massive membranes compared to other types of cells. In the present invention, MkMPs are first unloaded from the endogenous RNAs and then reloaded with the desirable RNAs, DNAs, proteins or other molecules for delivery to the target HSPCs. The present invention engineers cell-derived MkMPs where endogenous RNAs are removed from MPs using RNase treatment and exogenous molecules (plasmid DNA here) are loaded into MPs directly using electroporation. This process can be applied to other MPs beyond MkMPs and thus, we disclose for the first time a unique and powerful method for unloading the natural RNA cargo of MPs in order to re-load them with desirable “cargo”, i.e., any desirable molecules for delivery to target cells including HSPCs.
(109) In addition to view cell-derived MPs as a tool or a vesicle to deliver therapeutic drugs, MPs from specific cells (like endothelial cells, mesenchymal stem cells or other types) have unique biological function on target cells and other inventors used these MPs as a type of drugs to treat certain types of diseases. In U.S. patent application US 20120321723 A1, the inventors found MPs from stem cells, preferably a bone marrow-mesenchymal stem cell, a glomerular mesenchymal stem cell or a non-oval liver stem cell, exert anti-tumor effect when administered to a tumor patient.
BEST MODE FOR CARRYING OUT THE INVENTION
(110) 1. Source of Hematopoietic Stem and Progenitor Cells (HSPCs) and Generation of Megakaryocytes (
(111) Multiple sources of HSPCs, including but not limited to autologous or allogeneic CD34.sup.+ cells from bone marrow, peripheral blood or cord blood, are cultured and differentiated to megakaryocytes using a published protocol such as the one published in [9], but many other protocols and their variations can be used. Several such protocols have been disclosed in the scientific and patent literature. HSPCs can be also obtained from embryonic cells or from induced pluripotent stem (iPS) cells.
(112) 2. Generation of MkMPs, PLPs or PPTs from Mature or Maturing Megakaryocytes (
(113) To generate MkMPs or MPs from other cells, shear or other biomechanical forces are applied to the megakaryocytes to generate proplatelets/preplatelets (PPTs), platelet-like particles (PLPs) and megakaryocytic microparticles (MkMPs). The shear flow can be laminar or turbulent and flow application can be carried out in a parallel channel bioreactors, or mixed bioreactor where the cells are either freely suspended or growing on microcarriers (small beads of 100-1000 microns in diameter) where the cells can attach to for growth and maintenance. The intensity of biomechanical forces can be controlled by controlling the flow rate or the agitation/mixing rate as is known to someone skilled in the art, such as in described in Refs. [9-13]. The use of agitated or mixed bioreactors of freely suspended cells or cells attached to microcarriers for producing MPs is disclosed for the first time here. Microcarriers are small particles, mostly spherical but not only, whose surface is appropriately modified for allowing cells to attach and grow on the particle surface. [14, 15] Some microcarriers are also porous allowing cells to grow into the microcarriers. Both types of microcarriers are used for growing cells that require or prefer surface attachment and where the culture can be done both under static conditions but more typically in stirred bioreactors or vessels that enable scale up and quality control by controlling the culture conditions using various sensors such as for pH, dissolved oxygen, nutrient concentrations and metabolite concentrations. [12, 14-16] Tubular or channel bioreactors or microreactors or microfluidic reactors can be used to culture all types of cells and also to expose various human or mammalian cells to biomechanical forces. [13, 16-20] The use of such bioreactors for generating MPs or MVs or any other particle from mammalian or other cells is disclosed for the first time here. The use of such bioreactor systems for generating MPs or MVs or any other particle from mammalian or other cells could not have been anticipated by someone skilled in the art.
(114) 3. Separation, Purification and Storage of MkMPs, PLPs, and PPTs but Also MPs from Other Cell Types (
(115) Three types of particles, PPTs, PLPs and MkMPs, are collected and enriched from static cell culture and flow application using successive differential centrifugations as described in Ref. [9], but several other centrifugation, ultracentrifugation, elutriation, sedimentation and/or membrane protocols can be used as is well known by those skilled in the art [21-24]. MkMPs can be frozen as is known in the art of freezing hematopoietic stem cells for transplantation therapies. [25-27] PLPs or PPTs can either be stored at room temperature or used immediately. PLPs or PPTs can be also frozen using methods as for freezing MkMPs.
(116) 4. Modification of MkMPs for the Delivery of “Cargo” Molecules (DNA, RNA, Proteins, Etc) to HSPCs Both In Vivo and Ex Vivo (
(117) To engineer MkMPs, RNase is used to remove endogenous RNAs inside the microparticles by incubation of RNase and microparticles at 37° C. After that, the desirable RNAs, DNAs, proteins or other therapeutic drugs are loaded into MkMPs using electroporation (e.g., using Nucleofection performed on AMAXA Nucleofector® II Device, Lonza) or lipofection or other methods used in the modification of whole cells, but here applied to MkMPs.
(118) 5. Applications of the Produced Unmodified MkMPs, PLPs or PPTs (
(119) a. Hematopoietic transplantation for the reconstitution of HSPCs in vivo by intravenous infusion or co-infusion with HSPCs, either autologous or allogeneic. This is to enhance the in vivo expansion of HSPCs (by MkMPs), but also the in vivo megakaryopoiesis and platelet biogenesis (by MkMPs, PLPs, and PPTs). The latter two processes are beneficial to patients undergoing chemotherapy or patients with genetic or idiopathic disorders.
b. Ex vivo production of proplatelets or platelets. MkMPs also can be used as differentiation inducing reagent in ex vivo megakaryocytic differentiation and platelet production. The collected PPTs and PLPs can be used in clinical transfusion and intravenously infused to patients who need platelets, including those suffering severe thrombocytopenia disease, idiopathic or due to chemotherapy.
(120) 6. Applications of Modified MkMPs (
(121) The engineered MkMPs can be used in two different ways to deliver desirable materials (DNA, RNA, proteins, morphogens, or drugs) into target cells, including the HSPCs. One way is to infuse these engineered microparticles directly into the patient circulation. The other way is to coculture these microparticles with CD34+ HSPCs or other target cells ex vivo for 2-5 days and then transfuse those cells from coculture into patients.
(122) Although preferred embodiments of the disclosure are illustrated and described in connection with particular features, it will be apparent to those skilled in the art, that the invention can be adapted for use for a wide variety of applications. Various features of the disclosure have been particularly shown and described in connection with illustrated embodiments. However, it must be understood that the particular embodiments merely illustrate and that the invention is to be given its fullest interpretation within the terms of the claims.