Abstract
20 min readArticle Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Thrombocytopenic disorders have been treated with the Thrombopoietin-receptor agonist Eltrombopag. Patients with the same apparent form of thrombocytopenia may respond differently to the treatment. We describe a miniaturized bone marrow tissue model that provides a screening bioreactor for personalized, pre-treatment response prediction to Eltrombopag for individual patients. Using silk fibroin, a 3D bone marrow niche was developed that reproduces platelet biogenesis. Hematopoietic progenitors were isolated from a small amount of peripheral blood of patients with mutations in ANKRD26 and MYH9 genes, who had previously received Eltrombopag. The ex vivo response was strongly correlated with the in vivo platelet response. Induced Pluripotent Stem Cells (iPSCs) from one patient with mutated MYH9 differentiated into functional megakaryocytes that responded to Eltrombopag. Combining patient-derived cells and iPSCs with the 3D bone marrow model technology allows having a reproducible system for studying drug mechanisms and for individualized, pre-treatment selection of effective therapy in Inherited Thrombocytopenias. eLife digest Platelets are tiny cell fragments essential for blood to clot. They are created and released into the bloodstream by megakaryocytes, giant cells that live in the bone marrow. In certain genetic diseases, such as Inherited Thrombocytopenia, the bone marrow fails to produce enough platelets: this leaves patients extremely susceptible to bruising, bleeding, and poor clotting after an injury or surgery. Certain patients with Inherited Thrombocytopenia respond well to treatments designed to boost platelet production, but others do not. Why these differences exist could be investigated by designing new test systems that recreate the form and function of bone marrow in the laboratory. However, it is challenging to build the complex and poorly understood bone marrow environment outside of the body. Here, Di Buduo et al. have developed an artificial three-dimensional miniature organ bioreactor system that recreates the key features of bone marrow. In this system, megakaryocytes were grown from patient blood samples, and hooked up to a tissue scaffold made of silk. The cells were able to grow as if they were in their normal environment, and they could shed platelets into an artificial bloodstream. After treating megakaryocytes with drugs to stimulate platelet production, Di Buduo et al. found that the number of platelets recovered from the bioreactor could accurately predict which patients would respond to these drugs in the clinic. This new test system enables researchers to predict how a patient will respond to treatment, and to tailor therapy options to each individual. This technology could also be used to test new drugs for Inherited Thrombocytopenias and other blood-related diseases; if scaled-up, it could also, one day, generate large quantities of lab-grown blood cells for transfusion. Introduction Bone marrow megakaryocytes are responsible for the continuous production of platelets in the blood, driven by Thrombopoietin (TPO) through interaction with its receptor MPL (Hitchcock and Kaushansky, 2014; Kaushansky, 2015). In vivo, megakaryocytes associate with bone marrow microvasculature, where they extend proplatelets that protrude through the vascular endothelium into the lumen and release platelets into the bloodstream (Ito et al., 2018; Junt et al., 2007). Countless human pathologies result in alterations in platelet production; yet, for many of these, pathogenesis, and thus optimal targeted therapies, remain unknown. Inherited Thrombocytopenias are a diverse group of disorders characterized by low platelet count, resulting in impaired hemostasis. While often stable, patients can experience hemorrhages and/or excessive bleeding provoked by hemostatic events such as trauma or surgery; in some cases, hemorrhages appear spontaneously (Balduini et al., 2018; Balduini et al., 2017). The treatment of Inherited Thrombocytopenias is still unsatisfactory. For patients affected with the severe forms, which are usually fatal at young ages, the treatment of choice is hematopoietic stem cell transplantation (Balduini et al., 2013; Locatelli et al., 2003; Notarangelo et al., 2008). However, for most patients with Inherited Thrombocytopenias, transplantation is not recommended as the risks outweigh the benefits. The standard treatment protocols for these subjects were platelet transfusions to stop or prevent bleeding following trauma or during invasive procedures, anti-fibrinolytic agents, recombinant factor VIIa (rVIIa), or local treatment. A significant advance in the treatment of thrombocytopenias is the use of drugs that stimulate platelet production by mimicking the effects of TPO. The TPO-receptor agonists Eltrombopag, Romiplostim, and very recently Avatrombopag, have been approved for the treatment of several forms of acquired thrombocytopenia (Bussel, 2018; Cheng, 2011; Erickson-Miller et al., 2009; Kuter, 2013; Santini and Fenaux, 2015). TPO-receptor agonists were first explored in Inherited Thrombocytopenias in 2010 in a phase 2 trial of Eltrombopag in 12 patients with Myosin Heavy Chain 9 (MYH9) mutations (Pecci et al., 2010). In 2015, Eltrombopag was tested in eight patients with Wiskott-Aldrich syndrome with platelet increases primarily in the X-Linked Thrombocytopenia (XLT) patients (Gerrits et al., 2015). More recently, a follow on phase 2 trial showed that Eltrombopag was safe and effective in increasing platelet count and reducing bleeding symptoms in patients with different forms of Inherited Thrombocytopenia, including MYH9-Related Diseases (MYH9-RD), Ankyrin Repeat Domain 26-Related Thrombocytopenia (ANKRD26-RT), XLT/Wiskott-Aldrich syndrome, monoallelic Bernard-Soulier syndrome and Integrin beta 3 (ITGB3)-Related Thrombocytopenia (Zaninetti et al., 2020). Further, elective surgeries in MYH9-RD patients with severe thrombocytopenia have been performed safely after administration of Eltrombopag (Zaninetti et al., 2019). Overall, these studies indicated that a sizeable proportion of patients with Inherited Thrombocytopenia respond to Eltrombopag, but that the extent of platelet response is highly variable not only among different forms of Inherited Thrombocytopenia but also among different patients affected by the same disease. Tools that recapitulate the function of specific tissues or organs are critical to test drug efficacy, reduce ineffective or suboptimal therapies, and personalize the choice of the best treatment for each specific patient as exemplified by organoids. Reproduction of the bone marrow has been very difficult because of its incompletely understood complexity. Current research is focused on duplicating characteristic features of the physiologic bone marrow microenvironment ex vivo using relevant biomaterials and bioreactors, along with appropriate human cell sources (Chou et al., 2020; Di Buduo et al., 2018; Di Buduo et al., 2021). Silk is a naturally derived protein biomaterial with utility for studying platelet production since its fundamental features include non-thrombogenicity, low-immunogenicity, and non-toxicity (Abbonante et al., 2017; Di Buduo et al., 2017; Di Buduo et al., 2015; Omenetto and Kaplan, 2010). A combination of modular flow chambers and vascular silk tubes and sponges was used to record platelet generation by primary human megakaryocytes, in response to variations in surface stiffness, functionalization with extracellular matrix components, and co-culture with endothelial cells (Di Buduo et al., 2017; Di Buduo et al., 2015). These systems were able to support efficient platelet formation and, upon perfusion, recovery of functional platelets, as assessed through multiple activation tests, including participation in clot formation and thrombus formation under flow conditions (Di Buduo et al., 2017; Di Buduo et al., 2015). We developed an ex vivo miniaturized 3D bone marrow tissue model that recapitulates ex vivo platelet biogenesis of patients with different forms of Inherited Thrombocytopenias. This device is a radical improvement of the previous model because it minimizes the number of cultured cells required in an unlimited number of simultaneous culture chambers. The results, starting from only 15 mL of peripheral blood, showed that the ex vivo tissue model could predict the in vivo clinical platelet response to Eltrombopag in individual patients. The number of platelets recovered in the ex vivo model under standardized conditions, including exposure to Eltrombopag, was significantly correlated with the increase in platelet count observed in vivo after Eltrombopag treatment in the same patients. Overall, our data suggest this tissue model will have substantial applicability for the evaluation of the effects of compounds to determine their impact on platelet production. Results Device design and prototyping In adults, hematopoietic bone marrow is located in the medullary cavity of flat and long bones (Travlos, 2006), served by blood vessels that branch out into millions of small thin-walled arterioles and sinusoids allowing mature blood cells to enter the bloodstream (Figure 1A). To mimic such a structure, a device prototype of rectangular shape with 30 × 30 × 14 mm size and hollow cavities of 2 × 15 × 3.5 mm was developed. The device was connected to an outside peristaltic electronic pump (Figure 1A) through 0.9 mm diameter channels equipped with luer lock adaptors. We used devices with up to two reservoirs; however, the chamber can be designed to provide as many channels as required by the experimental conditions (Figure 1—figure supplement 1). Crosstalk between channels inside the device was eliminated by appropriate spatial separation and independent perfusion to allow assessment of patient-specific responses, following simultaneous exposure to TPO alone and TPO in combination with the tested drug. Figure 1 with 2 supplements see all Download asset Open asset Design of the bone marrow mimicking device. (A) To mimic the vascularized bone marrow tissue structure ex vivo a double-flow chamber device was designed in two parts. The core contains two separates flow channels dedicated to the perfusion having inlet and outlet ports for connection to a perfusion system. (B,C) The dimension of the polydimethylsiloxane (PDMS) mold cover top and (D,E) the core device is expressed in millimeters. Alternative models of the device are shown in Figure 1—figure supplement 1. The 3D-printed negative mold of the chamber is shown in Figure 1—figure supplement 2. 3D printing technology is one emerging option for producing new devices in a customized, fast, and cost-effective manner. The printing process for the negative mold of our device is easily scalable. It can be created in less than 1 hr using a polylactic acid (PLA High temperature, FormFutura Volcano, Figure 1—figure supplement 2), which allows casting and curing of polydimethylsiloxane (PDMS), a non-toxic polymeric organosilicon. The final shape of the system is optically clear (Figure 1B–E). Importantly, the device is reusable and autoclavable to ensure overall sterility to the system. Silk biomaterials for bone marrow system assembly and characterization A silk fibroin structure functionalized with fibronectin was prepared with salt leaching method and inserted into the device to model a spongy scaffold that reproduces bone marrow architecture, composition, and microcirculation (Figure 2A–C). A 2 days production process allowed us to obtain a sterile 3D silk-fibronectin scaffold that could be stored in water, at 4°C, up to 1 month after preparation and used upon experimental needs. The silk scaffold was connected to gas-permeable tubing allowing perfusion of the media with a peristaltic pump connected to inlet and outlet ports (Figure 2A). A cover cap closes the system before starting perfusion. The 3D reconstruction of the silk scaffold revealed the presence of multiple spatially distinct niches (Figure 2D and E) and also demonstrated the homogeneous distribution of pores from top to bottom of the scaffold (Figure 2F). This arrangement efficiently supported the diffusion of cells (Figure 2G) and media outflow without altering the shape and integrity of the silk. Importantly, the total volume collected after perfusion corresponded to that injected in the system by the pump. Figure 2 Download asset Open asset Silk sponge bone marrow perfusion system. (A–C) A peristaltic pump drives perfusion of the cell culture medium from a reservoir to the device equipped with a silk fibroin sponge prepared directly inside the chamber by dispensing an aqueous silk solution mixed with salt particles (scale bar B = 1.5 cm; scale bar C = 2 mm). After leaching out the salt, the resulting porous silk sponge can be sterilized. (D,E) Confocal microscopy reconstruction of the silk sponge showed the presence of an interconnected alveolar network (scale bar D = 200 µm; scale bar E = 150 µm). (F) The analysis of pore diameters measured on the top and bottom of the scaffold demonstrated no significant differences throughout the scaffold. Results are presented as mean ± SD (n = 150 pore/condition, p=NS). (G) Confocal microscopy analysis of CFSE+ cells cultured within the silk scaffold (red = CFSE; gray = silk; scale bar = 50 µm). The full data set is provided in Figure 2—source data 1. Figure 2—source data 1 Analysis of the pore diameter of the silk scaffolds. https://cdn.elifesciences.org/articles/58775/elife-58775-fig2-data1-v1.xlsx Download elife-58775-fig2-data1-v1.xlsx Tuning of the silk bone marrow device for testing hematopoietic progenitor response to drugs To ascertain the ability of the device to model physiological and pathological bone marrow, we took advantage of our expertise in culturing human hematopoietic stem and progenitor cells from peripheral blood of healthy controls and patients affected by two forms of Inherited Thrombocytopenia: ANKRD26-RT and MYH9-RD (Bluteau et al., 2014; Pecci et al., 2009). The bone marrow device was able to support efficient differentiation of mature megakaryocytes from both healthy controls and patients (Figure 3A and B). However patient-derived megakaryocytes displayed a decreased percentage of proplatelet formation by about 80%, accompanied by less branching of proplatelet shafts due to a significantly lower number of bifurcations (Healthy Control: 9 ± 2; ANKRD26-RT: 1.9 ± 0.7; MYH9-RD 1.8 ± 0.9) as compared to healthy controls (Figure 3C–E). Figure 3 with 1 supplement see all Download asset Open asset Modeling physiological and pathological megakaryopoiesis. (A) Megakaryocytes were differentiated from healthy controls and patients affected by MYH9-RD and ANKRD26-RT patients and cultured into the bone marrow device in presence of 10 ng/mL TPO. (B) Output of CD41+CD42b+ megakaryocyte at the end of differentiation relative to healthy controls (n = 12 Healthy Controls, n = 12 MYH9-RD; n = 12 ANKRD26-RT) (C) Percentage of proplatelet formation relative to healthy controls (n = 12 Healthy Controls, n = 12 MYH9-RD; n = 12 ANKRD26-RT; *p<0.01). (D) The number of proplatelet bifurcation per single megakaryocytes in healthy controls and patients (n = 12 Healthy Controls, n = 12 MYH9-RD; n = 12 ANKRD26-RT; *p<0.01). (E) Representative immunofluorescence staining of proplatelet structure (red=β1-tubulin; blue = nuclei; scale bar = 20 µm). All results are presented as mean ± SD. Data from the treatment of healthy controls in the presence of TPO and TPO +EPAG are shown in Figure 3—figure supplement 1. The full data set is provided in Figure 3—source data 1. Figure 3—source data 1 Analysis of megakaryocyte differentiation and proplatelet formation in healthy controls and patients. https://cdn.elifesciences.org/articles/58775/elife-58775-fig3-data1-v1.xlsx Download elife-58775-fig3-data1-v1.xlsx To validate the predictive value of the miniaturized bone marrow response to drugs specifically targeting hematopoiesis, we chose Eltrombopag as te model compound since Eltrombopag represents to date the only tested drug shown to increase platelet count of patients with Inherited Thrombocytopenias. First, we verified the ex vivo efficacy of Eltrombopag on human adult megakaryocytic progenitors from healthy controls and demonstrated the ability of the drug to increase megakaryocyte output and proplatelet formation with respect to the untreated control (Figure 3—figure supplement 1). Then, we tested the sensitivity of 24 pathological samples from ANKRD26-RT and MYH9-RD patients (Table 1). This cohort included 11 patients previously treated with Eltrombopag in a recent phase 2 clinical trial (Zaninetti et al., 2020) and two patients previously treated in preparation for elective surgery (Zaninetti et al., 2019). Blood samples for this study were collected when patients were out of Eltrombopag therapy and had platelet count at their baseline levels. Equal numbers of megakaryocytic progenitors were divided between each channel for the ex vivo culture. Table 1 Main features of the study population. ANKRD26-RTMYH9-RDTotal samples, no.1212M/F9/35/7Age - mean [range], years46 [22-67]48 [26-59]Platelet count - mean [range] x109/L32 [9-75]29 [5-69] Patients with Inherited Thrombocytopenias have normal or slightly increased serum levels of TPO (Zaninetti et al., 2020), thus, in vivo hematopoietic progenitors are exposed to stimuli from endogenous TPO simultaneously with Eltrombopag treatment. To mimic this condition faithfully, all the samples were cultured in the presence of 10 ng/mL recombinant human TPO alone or in combination with 500 ng/mL Eltrombopag (Figure 4A). Insights into the efficacy of Eltrombopag effects ex vivo were gained by simultaneously analyzing megakaryocyte differentiation at day 14 for each disorder. Specifically, cells were washed out of the device and analyzed. We observed comparable megakaryocyte maturation in terms of cell size (Figure 4B), ploidy profile (Figure 4C), and expression of lineage-specific markers (Figure 4D and E), with and without Eltrombopag. However, the combination of TPO and Eltrombopag resulted in a significant two-fold increase in the output of mature megakaryocytes with respect to TPO alone for both ANKRD26-RT and MYH9-RD patients (Figure 4F). Figure 4 Download asset Open asset Eltrombopag promotes megakaryocyte differentiation ex vivo. (A) Megakaryocytes were differentiated from peripheral blood progenitors of patients affected by MYH9-RD or ANKRD26-RT and cultured in the silk bone marrow tissue device in the presence of 10 ng/mL TPO supplemented or not with 500 ng/mL Eltrombopag (EPAG) and analyzed. The figure of the microscope was adapted from Servier Medical Art licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com). (B) Representative immunofluorescence staining of CD61 (red = CD61; blue = nuclei; scale bar = 25 µm) and (C) analysis of ploidy levels at the end of the culture (TPO: n = 3 MYH9-RD; n = 3 ANKRD26-RT; TPO +EPAG: n = 3 MYH9-RD; n = 3 ANKRD26-RT; p=NS). (D) Representative flow cytometry analysis of CD41+CD42b+ megakaryocytes at the end of the culture and (E) statistical analysis of mean fluorescence intensity (MFI) of the markers (TPO: n = 12 MYH9-RD; n = 12 ANKRD26-RT; TPO +EPAG: n = 12 MYH9-RD; n = 12 ANKRD26-RT; p=NS). (F) Output was calculated as the increase in the percentage of CD41+CD42b+ cells in presence of TPO +EPAG with respect to the percentage of cells in presence of TPO alone n = n = All results are presented as mean ± SD. The full data set is provided in Figure data 1. Figure data 1 Analysis of megakaryocyte Download of proplatelet formation to on mechanisms of Confocal microscopy analysis of 3D revealed a homogeneous distribution of megakaryocytes throughout the in both culture conditions, with in the presence of Eltrombopag, from both ANKRD26-RT and MYH9-RD (Figure Further, in the presence of Eltrombopag, megakaryocytes characteristic of proplatelets (Figure staining of megakaryocytes from the device and that TPO in combination with Eltrombopag supported the of multiple shafts platelets at their (Figure and a significant increase in the percentage of megakaryocytes in both ANKRD26-RT (TPO: 3 ± TPO ± and MYH9-RD (TPO: 1.5 ± TPO ± (Figure Figure Download asset Open asset Eltrombopag increased proplatelet formation ex vivo. (A) Confocal microscopy analysis of 3D megakaryocyte culture at the end of Megakaryocytes were proplatelet which platelets at their within the hollow of silk pores (red = blue = (scale = 50 µm). Analysis of proplatelet structure was performed by immunofluorescence staining of the (red=β1-tubulin; blue = nuclei; scale bar = 25 µm). In both diseases, the increased and branching of proplatelet shafts in presence of TPO +EPAG with respect to TPO (B) The percentage of proplatelet megakaryocytes was calculated as the number of cells long with respect to the total number of megakaryocytes per (TPO: n = 12 MYH9-RD; n = 12 ANKRD26-RT; TPO +EPAG: n = 12 MYH9-RD; n = 12 ANKRD26-RT; All results are presented as mean ± SD. The full data set is provided in Figure data 1. Figure data 1 Analysis of proplatelet Download vivo platelet count as a of drug efficacy the of Eltrombopag in patients with ANKRD26-RT or MYH9-RD is an increase in platelet count, platelet production was the most in our ex vivo we tested the to and count ex vivo platelets by the cultured with megakaryocytes from healthy controls in the presence of TPO alone or TPO in combination with Eltrombopag. day 15 of each channel of the device was connected to a peristaltic pump at the inlet and a gas-permeable at the The number of ex vivo platelets was assessed and with a standard by flow cytometry after 4 hr of perfusion, at and (Figure The mean number of collected platelets was 24 × × in the presence of with a significant increase in the presence of TPO in combination with Eltrombopag Figure Download asset Open asset vivo platelet count for response to (A) The flow chamber was with culture media and released platelets collected into gas-permeable before by flow (B) microscopy and analysis of the collected medium demonstrated the presence of large and platelets having the in platelets scale = 10 µm). (C) Representative flow cytometry analysis of expression of and surface (D) Analysis of the between the increase of platelet count ex vivo and the increase of platelet count observed in vivo from the same patients. For the ex vivo platelet count was calculated by flow cytometry with (n = MYH9-RD; n = 9 (E) Analysis of the between ex vivo megakaryocyte output and the increase of platelet count observed in vivo from the same patients. (n = MYH9-RD; n = 9 The full data set is provided in Figure data 1. Figure data 1 Analysis of platelet count and megakaryocyte output ex vivo, and with platelet count in vivo. Download To test our device could predict the patient-specific response to Eltrombopag, we performed a study the extent of platelet production ex vivo to the platelet response observed in vivo in the same patients (Zaninetti et al., et al., 2020). were in the same standardized conditions used with healthy After perfusion, ex vivo collected platelets the at their in peripheral blood platelets (Figure the physiological of the bone marrow environment for in vivo vivo collected platelets were with and and by flow cytometry (Figure The number of CD41+CD42b+ platelets collected per single channel increased significantly when treated with TPO in combination with Eltrombopag with respect to TPO in both ANKRD26-RT and MYH9-RD (Table However, all samples from healthy controls responded to the treatment with Eltrombopag, in patients the platelet response was with some samples a or no increase in ex vivo platelet production. The same was during the treatment in vivo (Zaninetti et al., et al., 2020). the increase in platelet count ex vivo in response to Eltrombopag was compared with the increase in platelet count observed in vivo after Eltrombopag administration in the same patients (Table 2), was a significant = (Figure The of this was supported by that the of the platelet count in vivo after Eltrombopag administration with the megakaryocyte output calculated ex vivo not the same = (Figure that ex vivo platelet count is the that is to predict the response Table 2 Main features of the study treated with Eltrombopag in vivo and ex vivo. treated with Eltrombopag in vivo and ex vivo, - mean [range], count at baseline mean [range], of platelet count after Eltrombopag treatment - mean [range], count TPO mean [range], of platelet count TPO mean [range], of one patient two ex vivo. of patients two ex vivo. of platelet count with Eltrombopag with respect to of platelet count with Eltrombopag with respect to the untreated Device with stem cells from patients at the genetic and levels is an in the of and to treatment. Induced stem cells (iPSCs) a to study mechanisms and testing were from one MYH9-RD patient and one healthy were tested for and found for and by analysis (Figure supplement 1A). and expression was also by immunofluorescence analysis (Figure supplement control and patient displayed a normal without (Figure supplement differentiation of was in culture conditions (Figure and Figure supplement and demonstrated that MYH9-RD iPSCs a in proplatelet formation ± 3.0 ± and branching of ± 1.5 ± with respect to control iPSCs (Figure Figure with 4 supplements see all Download asset Open asset of megakaryocyte (A) were cultured for days and by flow cytometry to megakaryocytes the mean fluorescence intensity (MFI) of MYH9-RD for and CD61 relative to healthy controls (n = 3 Healthy Controls, n = 3 MYH9-RD; p=NS). (B) Representative of proplatelet at day of culture from different (C) Percentage of proplatelet formation from the different (n = Healthy n = MYH9-RD two
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