Comparative Transcriptomic Analysis of GMP-Compliant Regulatory Macrophages (TRI-001) Under Hypoxic and Inflammatory Conditions
ASA Poster Gallery. Albrecht M. 10/11/25; 4177879; A1170
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Type: Experimental Circulation - ePosters Monitor 23
Topic: EXPERIMENTAL CIRCULATION - Ischemia and Reperfusion Injury
Martin Albrecht, PhD1, Rouven Berndt, MD2, Hess Katharina, MD3, Fred Faendrich, MD1, Tuija Kekarainen, PhD4, Lars Hummitzsch, MD1, Ole Sattler, MD1, Jens Scholz, MD, PhD1, Markus Steinfath, MD1, Karina Zitta, PhD1.
1University Medical Center Schleswig-Holstein, Kiel, Germany, 2University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 3HMU Health and Medical University, Potsdam, Germany, 4Ferring Ventures Oy, Kuopio, Finland.
Background:
Regulatory macrophages (Mreg) represent a unique subset of macrophages known for their anti-inflammatory and angiogenic properties, positioning them as promising candidates for cell-based therapies. Recently, we have differentiated and characterized a distinct Mreg subtype (TRI-001), which is currently being produced in accordance with good manufacturing practice (GMP) for a multicenter study aimed at treating patients with peripheral arterial occlusive disease (PAOD; clinical trial number: EudraCT 2024-517765-16).
Aim of the Study: To compare the transcriptome of TRI-001 with various in vitro differentiated macrophage subtypes to provide a comprehensive context for TRI-001 within the macrophage landscape. Additionally, we aimed to develop a detailed transcriptome profile of TRI-001 under transient hypoxic and inflammatory conditions, mimicking the microenvironment in PAOD patients.
Methods:
Mreg were differentiated from human CD14+ monocytes using a GMP-compliant protocol and identified as TRI-001 by flow cytometry. Hypoxia was induced via an enzymatic model, while inflammatory conditions were simulated using LPS treatment. Transcriptomic profiling was performed using Illumina HiSeq 4000 with 2 × 150 bp paired-end sequencing at a depth of 30 million reads. In vitro wound healing/migration assays were conducted using human umbilical vein endothelial cells (HUVEC) cultured with supernatants derived from normoxia and hypoxia treated TRI-001.
Results:
TRI-001 demonstrated significant transcriptomic similarities with Mreg and Mreg_UKR but were different from M0, M1, M2a, and PCMO subtypes (Figure I). Under hypoxic and inflammatory conditions, TRI-001 displayed distinct gene expression profiles compared to TRI-001 under control conditions, with hypoxic and LPS-stimulated profiles showing notable overlap (Figure II). Pathway enrichment analysis suggested the activation of chemotaxis and migration-associated pathways especially under hypoxic conditions.
Results:
from functional in vitro wound healing/migration assays were less conclusive, but suggest that the secretome from hypoxia- and normoxia-treated TRI-001 slightly increased migration of endothelial cells..
Conclusion:
TRI-001 represents a novel type of regulatory macrophages (Mreg). The distinctive transcriptional responses to hypoxia and inflammatory stimuli highlight its potential as a cell therapy for the treatment of PAOD patients.
Topic: EXPERIMENTAL CIRCULATION - Ischemia and Reperfusion Injury
Martin Albrecht, PhD1, Rouven Berndt, MD2, Hess Katharina, MD3, Fred Faendrich, MD1, Tuija Kekarainen, PhD4, Lars Hummitzsch, MD1, Ole Sattler, MD1, Jens Scholz, MD, PhD1, Markus Steinfath, MD1, Karina Zitta, PhD1.
1University Medical Center Schleswig-Holstein, Kiel, Germany, 2University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 3HMU Health and Medical University, Potsdam, Germany, 4Ferring Ventures Oy, Kuopio, Finland.
Background:
Regulatory macrophages (Mreg) represent a unique subset of macrophages known for their anti-inflammatory and angiogenic properties, positioning them as promising candidates for cell-based therapies. Recently, we have differentiated and characterized a distinct Mreg subtype (TRI-001), which is currently being produced in accordance with good manufacturing practice (GMP) for a multicenter study aimed at treating patients with peripheral arterial occlusive disease (PAOD; clinical trial number: EudraCT 2024-517765-16).
Aim of the Study: To compare the transcriptome of TRI-001 with various in vitro differentiated macrophage subtypes to provide a comprehensive context for TRI-001 within the macrophage landscape. Additionally, we aimed to develop a detailed transcriptome profile of TRI-001 under transient hypoxic and inflammatory conditions, mimicking the microenvironment in PAOD patients.
Methods:
Mreg were differentiated from human CD14+ monocytes using a GMP-compliant protocol and identified as TRI-001 by flow cytometry. Hypoxia was induced via an enzymatic model, while inflammatory conditions were simulated using LPS treatment. Transcriptomic profiling was performed using Illumina HiSeq 4000 with 2 × 150 bp paired-end sequencing at a depth of 30 million reads. In vitro wound healing/migration assays were conducted using human umbilical vein endothelial cells (HUVEC) cultured with supernatants derived from normoxia and hypoxia treated TRI-001.
Results:
TRI-001 demonstrated significant transcriptomic similarities with Mreg and Mreg_UKR but were different from M0, M1, M2a, and PCMO subtypes (Figure I). Under hypoxic and inflammatory conditions, TRI-001 displayed distinct gene expression profiles compared to TRI-001 under control conditions, with hypoxic and LPS-stimulated profiles showing notable overlap (Figure II). Pathway enrichment analysis suggested the activation of chemotaxis and migration-associated pathways especially under hypoxic conditions.
Results:
from functional in vitro wound healing/migration assays were less conclusive, but suggest that the secretome from hypoxia- and normoxia-treated TRI-001 slightly increased migration of endothelial cells..
Conclusion:
TRI-001 represents a novel type of regulatory macrophages (Mreg). The distinctive transcriptional responses to hypoxia and inflammatory stimuli highlight its potential as a cell therapy for the treatment of PAOD patients.
Type: Experimental Circulation - ePosters Monitor 23
Topic: EXPERIMENTAL CIRCULATION - Ischemia and Reperfusion Injury
Martin Albrecht, PhD1, Rouven Berndt, MD2, Hess Katharina, MD3, Fred Faendrich, MD1, Tuija Kekarainen, PhD4, Lars Hummitzsch, MD1, Ole Sattler, MD1, Jens Scholz, MD, PhD1, Markus Steinfath, MD1, Karina Zitta, PhD1.
1University Medical Center Schleswig-Holstein, Kiel, Germany, 2University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 3HMU Health and Medical University, Potsdam, Germany, 4Ferring Ventures Oy, Kuopio, Finland.
Background:
Regulatory macrophages (Mreg) represent a unique subset of macrophages known for their anti-inflammatory and angiogenic properties, positioning them as promising candidates for cell-based therapies. Recently, we have differentiated and characterized a distinct Mreg subtype (TRI-001), which is currently being produced in accordance with good manufacturing practice (GMP) for a multicenter study aimed at treating patients with peripheral arterial occlusive disease (PAOD; clinical trial number: EudraCT 2024-517765-16).
Aim of the Study: To compare the transcriptome of TRI-001 with various in vitro differentiated macrophage subtypes to provide a comprehensive context for TRI-001 within the macrophage landscape. Additionally, we aimed to develop a detailed transcriptome profile of TRI-001 under transient hypoxic and inflammatory conditions, mimicking the microenvironment in PAOD patients.
Methods:
Mreg were differentiated from human CD14+ monocytes using a GMP-compliant protocol and identified as TRI-001 by flow cytometry. Hypoxia was induced via an enzymatic model, while inflammatory conditions were simulated using LPS treatment. Transcriptomic profiling was performed using Illumina HiSeq 4000 with 2 × 150 bp paired-end sequencing at a depth of 30 million reads. In vitro wound healing/migration assays were conducted using human umbilical vein endothelial cells (HUVEC) cultured with supernatants derived from normoxia and hypoxia treated TRI-001.
Results:
TRI-001 demonstrated significant transcriptomic similarities with Mreg and Mreg_UKR but were different from M0, M1, M2a, and PCMO subtypes (Figure I). Under hypoxic and inflammatory conditions, TRI-001 displayed distinct gene expression profiles compared to TRI-001 under control conditions, with hypoxic and LPS-stimulated profiles showing notable overlap (Figure II). Pathway enrichment analysis suggested the activation of chemotaxis and migration-associated pathways especially under hypoxic conditions.
Results:
from functional in vitro wound healing/migration assays were less conclusive, but suggest that the secretome from hypoxia- and normoxia-treated TRI-001 slightly increased migration of endothelial cells..
Conclusion:
TRI-001 represents a novel type of regulatory macrophages (Mreg). The distinctive transcriptional responses to hypoxia and inflammatory stimuli highlight its potential as a cell therapy for the treatment of PAOD patients.
Topic: EXPERIMENTAL CIRCULATION - Ischemia and Reperfusion Injury
Martin Albrecht, PhD1, Rouven Berndt, MD2, Hess Katharina, MD3, Fred Faendrich, MD1, Tuija Kekarainen, PhD4, Lars Hummitzsch, MD1, Ole Sattler, MD1, Jens Scholz, MD, PhD1, Markus Steinfath, MD1, Karina Zitta, PhD1.
1University Medical Center Schleswig-Holstein, Kiel, Germany, 2University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 3HMU Health and Medical University, Potsdam, Germany, 4Ferring Ventures Oy, Kuopio, Finland.
Background:
Regulatory macrophages (Mreg) represent a unique subset of macrophages known for their anti-inflammatory and angiogenic properties, positioning them as promising candidates for cell-based therapies. Recently, we have differentiated and characterized a distinct Mreg subtype (TRI-001), which is currently being produced in accordance with good manufacturing practice (GMP) for a multicenter study aimed at treating patients with peripheral arterial occlusive disease (PAOD; clinical trial number: EudraCT 2024-517765-16).
Aim of the Study: To compare the transcriptome of TRI-001 with various in vitro differentiated macrophage subtypes to provide a comprehensive context for TRI-001 within the macrophage landscape. Additionally, we aimed to develop a detailed transcriptome profile of TRI-001 under transient hypoxic and inflammatory conditions, mimicking the microenvironment in PAOD patients.
Methods:
Mreg were differentiated from human CD14+ monocytes using a GMP-compliant protocol and identified as TRI-001 by flow cytometry. Hypoxia was induced via an enzymatic model, while inflammatory conditions were simulated using LPS treatment. Transcriptomic profiling was performed using Illumina HiSeq 4000 with 2 × 150 bp paired-end sequencing at a depth of 30 million reads. In vitro wound healing/migration assays were conducted using human umbilical vein endothelial cells (HUVEC) cultured with supernatants derived from normoxia and hypoxia treated TRI-001.
Results:
TRI-001 demonstrated significant transcriptomic similarities with Mreg and Mreg_UKR but were different from M0, M1, M2a, and PCMO subtypes (Figure I). Under hypoxic and inflammatory conditions, TRI-001 displayed distinct gene expression profiles compared to TRI-001 under control conditions, with hypoxic and LPS-stimulated profiles showing notable overlap (Figure II). Pathway enrichment analysis suggested the activation of chemotaxis and migration-associated pathways especially under hypoxic conditions.
Results:
from functional in vitro wound healing/migration assays were less conclusive, but suggest that the secretome from hypoxia- and normoxia-treated TRI-001 slightly increased migration of endothelial cells..
Conclusion:
TRI-001 represents a novel type of regulatory macrophages (Mreg). The distinctive transcriptional responses to hypoxia and inflammatory stimuli highlight its potential as a cell therapy for the treatment of PAOD patients.
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