Corrigendum: Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.
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ABSTRACT:
SUBMITTER: Moreau T
PROVIDER: S-EPMC5537628 | biostudies-other | 2017 Jul
REPOSITORIES: biostudies-other
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Project description:The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.
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Project description:Microarray gene expression data is used to compare the transcriptomics of hiPSC, hiPSC derived forward programmed-, hiPSC derived directed differentiated- megakaryoctes AND cord- blood derived megakaryocytes
2016-03-01 | GSE54822 | GEO
Project description:Factors limiting the adoption of iPSC technology include the cost of developing lines and the time period that it takes to characterize and bank them, particularly when integration free, feeder free, and Xeno-free components are used. In this manuscript we describe our optimization procedure that enables a single technician to make 20-40 lines at a time in a 24-96 well format in a reliable and reproducible fashion. Improvements spanned the entire workflow and included using RNA virus, reducing cytotoxicity of reagents, developing improved transfection and freezing efficiencies, modifying the manual colony picking steps, enhancing passaging efficiency and developing early criteria of success. These modifications allowed us to make more than two hundred well-characterized lines per year.
| S-EPMC4464084 | biostudies-other
Project description:The production of in vitro-derived platelets has great potential for transfusion medicine. Here, we build on our experience in the forward programming (FoP) of human pluripotent stem cells (hPSCs) to megakaryocytes (MKs) and address several aspects of the complex challenges to bring this technology to the bedside. We first identify clinical-grade hPSC lines that generate MKs efficiently. We design a bespoke media to maximize both production and maturity of MKs and improve platelet output. Crucially, we transition the lentiviral-based FoP of hPSCs to a nonviral inducible system. We also show how small molecules promote a definitive hematopoiesis phenotype during the differentiation process, thereby increasing the quality of the final product. Finally, we generate platelets using a bioreactor designed to reproduce the physical cues that promote platelet production in the bone marrow. We show that these platelets are able to contribute to both thrombus formation in vitro and have a hemostatic effect in thrombocytopenic mice in vivo.
| S-EPMC8045491 | biostudies-literature
Project description:Megakaryocytes (MKs) are the precursors of platelets (PLTs) and may be used for PLT production in vivo or in vitro, as well as a source for PLT-derived growth factors. Induced pluripotent stem cells represent an unlimited cell source for the in vitro production of MKs. This study aimed at developing an effective, xeno-free and scalable system to produce high numbers of MKs. In particular, microcarrier beads-assisted stirred bioreactors were evaluated as a means of improving MK yields. This method resulted in the production of 18.7?×?107 MKs per 50?ml medium. Laminin-coated microcarriers increased MK production per iPSC by up to 10-fold. MKs obtained in this system showed typical features of mature MKs and were able to produce PLTs in vitro and in vivo. To increase safety, MKs produced in the bioreactors were irradiated; a procedure that did not affect their capability to form proPLTs and PTLs after transfusion. In vitro generated MKs represent a promising alternative to donor PLTs and open the possibility for the development of innovative MK-based cell therapies.
| S-EPMC6033877 | biostudies-other
Project description:Platelet deficiency, known as thrombocytopenia, can cause hemorrhage and is treated with platelet transfusions. We developed a system for the production of platelet precursor cells, megakaryocytes, from pluripotent stem cells. These cultures can be maintained for >100 days, implying culture renewal by megakaryocyte progenitors (MKPs). However, it is unclear whether the MKP state in vitro mirrors the state in vivo, and MKPs cannot be purified using conventional surface markers. We performed single-cell RNA sequencing throughout in vitro differentiation and mapped each state to its equivalent in vivo. This enabled the identification of five surface markers that reproducibly purify MKPs, allowing us insight into their transcriptional and epigenetic profiles. Last, we performed culture optimization, increasing MKP production. Together, this study has mapped parallels between the MKP states in vivo and in vitro and allowed the purification of MKPs, accelerating the progress of in vitro-derived transfusion products toward the clinic.
| S-EPMC8849335 | biostudies-literature
Project description:ObjectivesEx vivo engineered production of megakaryocytes (MKs) and platelets (PLTs) from human pluripotent stem cells is an alternative approach to solve shortage of donor-donated PLTs in clinics and to provide induced PLTs for transfusion. However, low production yields are observed and the generation of clinically applicable MKs and PLTs from human pluripotent stem cells without genetic modifications still needs to be improved.Materials and methodsWe defined an optimal, stepwise and completely xeno-free culture protocol for the generation of MKs from human embryonic stem cells (hESCs). To generate MKs from hESCs on a large scale, we improved the monolayer induction manner to define three-dimensional (3D) and sphere-like differentiation systems for MKs by using a special polystyrene CellSTACK culture chamber.ResultsThe 3D manufacturing system could efficiently generate large numbers of MKs from hESCs within 16-18 days of continuous culturing. Each CellSTACK culture chamber could collect on an average 3.4 × 108 CD41+ MKs after a three-stage orderly induction process. MKs obtained from hESCs via 3D induction showed significant secretion of IL-8, thrombospondin-1 and MMP9. The induced cells derived from hESCs in our culture system were shown to have the characteristics of MKs as well as the function to form proPLTs and release PLTs. Furthermore, we generated clinically applicable MKs from clinical-grade hESC lines and confirmed the biosafety of these cells.ConclusionsWe developed a simple, stepwise, 3D and completely xeno-free/feeder-free/transgene-free induction system for the generation of MKs from hESCs. hESC-derived MKs were shown to have typical MK characteristics and PLT formation ability. This study further enhances the clinical applications of MKs or PLTs derived from pluripotent stem cells.
| S-EPMC8016648 | biostudies-literature
Project description:Patients suffering from acute or sustained thrombocytopenia require platelet transfusions, which are entirely donor-based and limited by challenges related to storage and fluctuating supply. Developing cell-culture technologies will enable ex vivo and donor-independent platelet production. However, critical advancements are needed to improve scalability and increase megakaryocyte (Mk) culture productivity. To address these needs, we evaluated Mk production from mobilized peripheral blood CD34+ cells cultured on a commercially available gas-permeable silicone rubber membrane, which provides efficient gas exchange, and investigated the use of fed-batch media dilution schemes. Starting with a cell-surface density of 40 × 103 CD34+ cells per cm2 (G40D), culturing cells on the membrane for the first 5 days and employing media dilutions yielded 39 ± 19 CD41+ CD42b+ Mks per input CD34+ cell by day 11-a 2.2-fold increase compared with using standard culture surfaces and full media exchanges. By day 7, G40D conditions generated 1.5-fold more CD34+ cells and nearly doubled the numbers of Mk progenitors. The increased number of Mk progenitors coupled with media dilutions, potentially due to the retention of interleukin (IL)-3, increased Mk production in G40D. Compared with controls, G40D had higher viability, yielded threefold more Mks per milliliter of media used and exhibited lower mean ploidy, but had higher numbers of high-ploidy Mks. Finally, G40D-Mks produced proplatelets and platelet-like-particles that activate and aggregate upon stimulation. These results highlight distinct improvements in Mk cell-culture and demonstrate how new technologies and techniques are needed to enable clinically relevant production of Mks for platelet generation and cell-based therapies.
| S-EPMC6591548 | biostudies-literature
Project description:Fetal stem cells are a unique type of adult stem cells that have been suggested to be broadly multipotent with some features of pluripotency. Their clinical potential has been documented but their upgrade to full pluripotency could open up a wide range of cell-based therapies particularly suited for pediatric tissue engineering, longitudinal studies or disease modeling. Here we describe episomal reprogramming of mesenchymal stem cells from the human amnion to pluripotency (AM-iPSC) in chemically defined conditions. The AM-iPSC expressed markers of embryonic stem cells, readily formed teratomas with tissues of all three germ layers present and had a normal karyotype after around 40 passages in culture. We employed novel computational methods to determine the degree of pluripotency from microarray and RNA sequencing data in these novel lines alongside an iPSC and ESC control and found that all lines were deemed pluripotent, however, with variable scores. Differential expression analysis then identified several groups of genes that potentially regulate this variability in lines within the boundaries of pluripotency, including metallothionein proteins. By further studying this variability, characteristics relevant to cell-based therapies, like differentiation propensity, could be uncovered and predicted in the pluripotent stage.
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Project description:Kidney organoids represent a physiologically advanced model for studying the mechanisms of kidney development and disease. Here, we describe a simple two-step protocol for the differentiation of human pluripotent stem cells into kidney organoids. Our approach involves suspension culture that allows for rapid and cost-effective bulk production of organoids, which is well suited for large-scale assays such as drug screening. The organoids correspond to fetal human kidney tissue and may be of limited use for modeling adult kidney function. For complete details on the use and execution of this protocol, please refer to Przepiorski et al. (2018).
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