<meta charset="utf8"></head><body>iPSC'den Türetilmiş Organoidlerle Timik Mikro Ortamın Yeniden Oluşturulması

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+human+thymic-derived+cells+display+medullary+thymic+epithelial+cell+phenotype+and+functionality.">Cultured human thymic-derived cells display medullary thymic epithelial cell phenotype and functionality.</a> Front Immunol. 9, 1663 (2018).</li><li>Hauri-Hohl, M., Zuklys, S., Holl&#228;nder, G. A., Ziegler, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+role+for+TGF-&#946;+signaling+in+the+establishment+and+function+of+the+thymic+medulla.">A regulatory role for TGF-&#946; signaling in the establishment and function of the thymic medulla.</a> Nat Immunol. 15 (6), 554-561 (2014).</li><li>Campinoti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+a+functional+human+thymus+by+postnatal+stromal+progenitor+cells+and+natural+whole-organ+scaffolds.">Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.</a> Nat Commun. 11 (1), 6372 (2020).</li><li>Ramos, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+thymic+organoids+from+human+pluripotent+stem+cells.">Generation of functional thymic organoids from human pluripotent stem cells.</a> Stem Cell Reports. 18 (4), 829-840 (2023).</li><li>Fan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineering+thymus+organoids+to+restore+thymic+function+and+induce+donor-specific+immune+tolerance+to+allografts.">Bioengineering thymus organoids to restore thymic function and induce donor-specific immune tolerance to allografts.</a> Mol Ther. 23 (7), 1262-1277 (2015).</li><li>Provin, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatory+differentiation+of+human+induced+pluripotent+stem+cells+generates+thymic+epithelium+that+supports+thymic+crosstalk+and+directs+dendritic-+and+CD4/CD8+T-cell+full+development.">Combinatory differentiation of human induced pluripotent stem cells generates thymic epithelium that supports thymic crosstalk and directs dendritic- and CD4/CD8 T-cell full development.</a> bioRxiv. 2023, 572664 (2023).</li><li>Parent, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+thymic+epithelium+from+human+embryonic+stem+cells+that+supports+host+T+cell+development.">Generation of functional thymic epithelium from human embryonic stem cells that supports host T cell development.</a> Cell Stem Cell. 13 (2), 219-229 (2013).</li><li>Sun, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+embryonic+stem+cells+into+thymic+epithelial+progenitor-like+cells+reconstitutes+the+thymic+microenvironment+in+vivo.">Directed differentiation of human embryonic stem cells into thymic epithelial progenitor-like cells reconstitutes the thymic microenvironment in vivo.</a> Cell Stem Cell. 13 (2), 230-236 (2013).</li><li>Inami, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+induced+pluripotent+stem+cells+to+thymic+epithelial+cells+by+phenotype.">Differentiation of induced pluripotent stem cells to thymic epithelial cells by phenotype.</a> Immunol Cell Biol. 89 (2), 314-321 (2011).</li><li>Gras-Pena, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cell-derived+thymic+epithelial+cells+enhance+human+T+cell+development+in+a+xenogeneic+thymus.">Human stem cell-derived thymic epithelial cells enhance human T cell development in a xenogeneic thymus.</a> J Allergy Clin Immunol. 149 (5), 1755-1771 (2022).</li><li>Lai, L., Jin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+thymic+epithelial+cell+progenitors+by+mouse+embryonic+stem+cells.">Generation of thymic epithelial cell progenitors by mouse embryonic stem cells.</a> Stem Cells. 27 (12), 3012-3020 (2009).</li><li>Ramos, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+human+thymic+cells+from+induced+pluripotent+stem+cells.">Generation of functional human thymic cells from induced pluripotent stem cells.</a> J Allergy Clin Immunol. 149 (2), 767-781 (2022).</li><li>Provin, N., Giraud, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+pluripotent+stem+cells+into+thymic+epithelial+cells+and+generation+of+thymic+organoids:+Applications+for+therapeutic+strategies+against+APECED.">Differentiation of pluripotent stem cells into thymic epithelial cells and generation of thymic organoids: Applications for therapeutic strategies against APECED.</a> Front Immunol. 13, 930963 (2022).</li><li>Montel-Hagen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+recapitulation+of+murine+thymopoiesis+from+single+hematopoietic+stem+cells.">In vitro recapitulation of murine thymopoiesis from single hematopoietic stem cells.</a> Cell Rep. 33 (4), 108320 (2020).</li><li>Park, J. -. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+atlas+of+human+thymic+development+defines+T+cell+repertoire+formation.">A cell atlas of human thymic development defines T cell repertoire formation.</a> Science. 367 (6480), 3224 (2020).</li><li>Flippe, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+reproducible+differentiation+of+hematopoietic+and+T+cell+progenitors+from+pluripotent+stem+cells.">Rapid and reproducible differentiation of hematopoietic and T cell progenitors from pluripotent stem cells.</a> Front Cell Dev Biol. 8, 577464 (2020).</li><li>Padonou, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aire-dependent+transcripts+escape+Raver2-induced+splice-event+inclusion+in+the+thymic+epithelium.">Aire-dependent transcripts escape Raver2-induced splice-event inclusion in the thymic epithelium.</a> EMBO Rep. 23 (3), e53576 (2022).</li><li>Han, J., Z&#250;&#241;iga-Pfl&#252;cker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-oxygen+submersion+fetal+thymus+organ+cultures+enable+FOXN1-dependent+and+-independent+support+of+T+lymphopoiesis.">High-oxygen submersion fetal thymus organ cultures enable FOXN1-dependent and -independent support of T lymphopoiesis.</a> Front Immunol. 12, 652665 (2021).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma Aldrich</td>
			<td>616370</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>Miltenyi</td>
			<td>130-111-165</td>
			<td></td>
		</tr>
		<tr>
			<td>CCR7 (CD197)</td>
			<td>BD Biosciences</td>
			<td>PE</td>
			<td>Clone: 3D12; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>BD Biosciences</td>
			<td>FITC</td>
			<td>Clone: M5E2; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>BD Biosciences</td>
			<td>PE</td>
			<td>Clone: HIB19; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD205</td>
			<td>BioLegend</td>
			<td>FITC</td>
			<td>Clone: MG38; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>BD Biosciences</td>
			<td>PE</td>
			<td>Clone: HIT3a; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD34</td>
			<td>BD Biosciences</td>
			<td>FITC</td>
			<td>Clone: 8G12; Dilution: 1: 100</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>BD Biosciences</td>
			<td>PE</td>
			<td>Clone: RPA-T4; Dilution: 1: 100</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>BD Biosciences</td>
			<td>BV711</td>
			<td>Clone: L200; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>BD Biosciences</td>
			<td>PerCP</td>
			<td>Clone: HI30; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD56</td>
			<td>BD Biosciences</td>
			<td>PE</td>
			<td>Clone: B159; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD62L</td>
			<td>BD Biosciences</td>
			<td>BV605</td>
			<td>Clone: DREG-56; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD69</td>
			<td>BD Biosciences</td>
			<td>BV510</td>
			<td>Clone: FN50; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD7</td>
			<td>BD Biosciences</td>
			<td>APC</td>
			<td>Clone: M-T701; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>BD Biosciences</td>
			<td>PeCy7</td>
			<td>Clone: RPA-T8; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>BD Biosciences</td>
			<td>PE</td>
			<td>Clone: HIT8a; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>Dynabeads Pan Mouse IgG</td>
			<td>Invitrogen</td>
			<td>11041</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Miltenyi</td>
			<td>130-097-751</td>
			<td></td>
		</tr>
		<tr>
			<td>EPCAM (CD326)</td>
			<td>BD Biosciences</td>
			<td>PE</td>
			<td>Clone: HEA-125; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>EPCAM (CD326)</td>
			<td>Miltenyi</td>
			<td>BV711</td>
			<td>Clone: EBA-1; Dilution: 1: 200</td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>Miltenyi</td>
			<td>130-127-858</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF8</td>
			<td>Biotechne R&#38;D</td>
			<td>423-F8</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma Aldrich</td>
			<td>341578</td>
			<td></td>
		</tr>
		<tr>
			<td>FLT3 L</td>
			<td>Peprotech</td>
			<td>AF-300-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-61</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF1</td>
			<td>Miltenyi</td>
			<td>130-093-886</td>
			<td></td>
		</tr>
		<tr>
			<td>IL7</td>
			<td>Peprotech</td>
			<td>AF-200-07</td>
			<td></td>
		</tr>
		<tr>
			<td>RANK L</td>
			<td>Biotechne R&#38;D</td>
			<td>6449-TEC</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis solution</td>
			<td>Miltenyi</td>
			<td>130-094-183</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>Peprotech</td>
			<td>AF-300-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma Aldrich</td>
			<td>605190</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>2605010</td>
			<td></td>
		</tr>
		<tr>
			<td>XVIVO10</td>
			<td>Lonza</td>
			<td>LONBE04-380Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

formation of human thymus organoids in three-dimensional fibrin hydrogels

The thymus is a primary lymphoid organ that plays an essential role in the generation of a competent and tolerant immune system1,2,3. Early thymic progenitors (ETPs) migrate from the bone marrow to the thymus, where they expand and differentiate into functional T cells1,2,4,5. Those processes are mediated by a specialized population, the thymic epithelial cells (TECs)2,6,7. TECs derive from thymic epithelial progenitors (TEPs)8,9 and comprise cortical TECs (cTECs) and medullary TECs (mTECs) that play specific roles in creating the specialized 3D microenvironment needed for T cell migration, expansion, and maturation. TECs mediate T cell development mainly by providing growth and differentiation factors1,10,11 and by negatively selecting non-functional and non-tolerant thymocytes through the presentation of self-antigens5,7,12. The complex interactions between developing T cells and TECs also play a central role in the maturation and 3D organization of the TEC populations in a process known as thymic crosstalk1,11. Interactions between the cell populations of the thymus rely deeply on the specific microenvironment shaped by the extracellular matrix (ECM). The thymic ECM is in a state of dynamic reciprocity with thymic cell populations, impacting gene regulation and being constantly reshaped in return by the secretion of enzymes or matrix proteins13. The ECM influences cells through modification of the bioavailability of growth factors and cytokines, direct signalization through membrane-bound receptors such as integrins, and by shaping cytoskeletons through physical forces14. Thymic ECM components, such as collagens and laminin, have been shown to have a high affinity for growth factors TGFb and FGFs, which are crucial for TEC maintenance and to fix&#160;them by forming complexes. Thymic ECM plasticity, elastic modulus, and density also play a crucial role in instructing TEC fate and shaping the compartmentalization of the thymus, which is essential to its functionality. These clues highlight the importance of taking into account the ECM and its 3D structure to mimic the thymus ex vivo. This point is supported by the fact that primary TECs quickly dedifferentiate, lose their functionality, and eventually die when cultivated in classical cell culture setups15,16,17.
Culture models have been developed to expand functional TEC populations from human thymic explants in order to conserve the structure of the ECM and the crucial clues it provides to TECs18,19,20. This culture system was able to successfully expand and maintain a population of functional TECs in vitro but could not be sustained past 7 to 8 days of culture18. Thus, the development of an accessible, practical 3D culture system capable of reproducing the thymic microenvironment and its functionality in vitro and in the long term is a crucial stake in the field. Recently, the development of hydrogel-based 3D culture systems has led to the emergence of several artificial thymic organoid systems, constituting major progress for in vitro thymic modelization15,16,21,22. We developed a human thymic organoid (hTO) co-culture system through the reaggregation of human primary ETPs with human TEPs derived from induced pluripotent stem cells (iPSC) into spheroids and their seeding upon a fibrin hydrogel.
The choice of material and hydrogel setup in this study aimed to reproduce the native structure of the thymic ECM while maintaining practicality and the ability to scale up the process to obtain an affordable and abundant material source for experiments15. This hTO system shows multilineage differentiation potential and can support a productive thymopoiesis from ETPs23. This organoid system constitutes a reliable tool for the study of intrathymic cellular interactions and the modeling of normal and pathological human lymphopoiesis. The use of iPS cells also introduces gene-editing capabilities into the model. Effective differentiation of iPSC into functional thymic tissue has been a longstanding goal of the field for the last 15 years, and significant progress has been made with deciphering TEC lineage fate signalling21,24,25,26,27.To answer the need for such an in vitro 3D thymic model, this technical note describes the methods and technical details for the step-by-step generation of iPSC-derived human thymus organoids, focusing on hydrogel scaffold formation, cell micromass reaggregation and seeding, and organoid culture and harvest.

<meta charset="utf8"></head><body>Yazar Spotlight: İnsan Timik Organoidleri ile Timik Epitel Hücrelerini ve T-Hücresi Araştırmalarını İlerletmek

Üç Boyutlu Fibrin Hidrojellerinde İnsan Timus Organoidlerinin Oluşumu

We are trying to study the mechanisms responsible for self-tolerance, autoimmunity, especially the role played by air in the expression of self-antigens presented by thymic epithelial cells to developing thymocytes. Our research has applications for cell therapy in the context of autoimmune diseases, organ transplants, and cancer. Our system produces thymic organoids, which are formed and seeded in a way that reproduces crucial biological and mechanical cues, which are essential for the maintenance and maturation of thymic epithelial cells.The mTECs, which in turn are essential to support thymopoiesis, can be maintained for up to six weeks in the system. Our findings contribute to developing better techniques for in vitro cell lymphocyte production with many potential applications for research and cell therapy. As our research progresses, we have to contribute to a better understanding of the mechanisms underlying self-tolerance and autoimmunity.We are currently focusing on characterizing our organoids further in terms of cellular composition and organization. And our goal is to explore the role played by the engine in regulating self-antigen expression in the thymus, and to establish a study model for the autoimmune disease episode.

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

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Research

JoVE Journal

Developmental Biology

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

963 Görüntüleme.  Nantes Université, CHU Nantes, INSERM, Center for Research in Transplantation and Translational Immunology, UMR 1064. Öz tolerans, otoimmünite, özellikle timik epitel hücreleri tarafından sunulan öz antijenlerin gelişmekte olan timositlere ekspresyonunda havanın oynadığı rolden sorumlu mekanizmaları incelemeye çalışıyoruz. Araştırmamızın otoimmün hastalıklar, organ nakli ve kanser bağlamında hücre tedavisi için uygulamaları vardır. Sistemimiz, timik epitel hücrelerinin bakımı ve olgunlaşması için gerekli olan önemli biyolojik ve mekanik ipu...

Video: Yazar Spotlight: İnsan Timik Organoidleri ile Timik Epitel Hücrelerini ve T-Hücresi Araştırmalarını İlerletmek

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

Here, we describe a protocol for the formation of human iPSC-derived thymus organoids grown in 3D fibrin hydrogels aiming to support thymic epithelial cell (TEC) maturation and prolonged maintenance, as well as thymopoiesis in vitro.

Gelişim Biyolojisi

3D Human Thymic Organoid Model Preparation for Studying T-Cell Biology

To begin, thaw the thrombin and aprotinin on ice, and place a fibrinogen vial in a 37 degree Celsius water bath. Using a pipette, homogenize them under the hood. Then using sterile pliers, place the inserts into the culture wells, leaving at least one column or row empty on the plate.To a 0.25 milliliter tube, first add the required volumes of fibrinogen and aprotinin. Next, add the thrombin into the tube and quickly flush two times without creating bubbles. Draw the entire content of the tube.Position the pipette vertically above the center of the insert and gently flush a reagent mix without generating bubbles. Incubate for at least one hour at 37 degrees Celsius for solidification until the mix turns opaque white. For organoid seeding, confirm under a microscope that the micro masses forms spheric cell masses, with a compact chorus surrounded by a lower density halo of early thymic progenitors.Cut the tip of a P200 cone and wash it with an anti-adherent solution. Tilt the plate to a nearly vertical position and aspirate the cell masses from the bottom of the well. Delicately deposit the mass at the top of the hydrogels without touching the gel.Check under the microscope that no organoid is left in the plate wells. Then slowly add a quarter of the volume of culture medium at the top of the hydrogel without touching it, and add the remaining 3/4 at the bottom of the well. Add one milliliter of PBS to the empty culture wells to maintain humidity in the plate and incubate at 37 degrees Celsius with 5%carbon dioxide.The organoids form spheric to oblong structures and occasionally merge to form larger structures in the hydrogels.