Employing epigenetic memory and native instructive stimuli to stimulate iPS-NLC differentiation

Notochordal cells (NCs) are linked to a healthy intervertebral disc (IVD), and are considered a promising candidate for cell-based therapies. However, NCs are scarcely available as they are lost early in life, and attempts at in vitro expansion have failed because NCs lose their specific phenotype. The production of notochordal-like cells (NLCs) from human induced pluripotent stem cells (hiPSCs) is a viable alternative. Therefore, this study aimed to build on the tissue-specific epigenetic memory of hiPSCs derived from IVD-progenitor cells (TIE2+-cells) and the instructive capacity of decellularized notochordal cell-derived matrix (dNCM)2 to improve hiPSC differentiation towards mature, healthy matrix-producing NLCs. hiPSCs were generated from TIE2+-IVD cells of three adult donors. As a comparison donor-matched minimally invasive peripheral blood mononuclear (PBM)-derived iPSCs were used. Firstly, hiPSCs were differentiated into mesendodermal progenitors by Wnt pathway activation (N2B27 medium + 3µM CHIR99021)1 for 2 days. Thereafter, the cells were further driven towards the NC-lineage by transfection with synthetic NOTO mRNA1 and matured by switching to a 3D-cell pellet culture in discogenic medium containing 10ng/mL TGF-β1 or 3mg/mL dNCM until day 28. Read-outs included cell morphology, gene and protein expression and matrix deposition. Both TIE2+- and PBM-cell derived hiPSC showed successful differentiation towards mesendodermal progenitors following Wnt-activation on day 2, indicated by the cells moving out of the colonies after CHIR stimulation. Accordingly, a decreased gene expression of pluripotency markers (OCT4, SOX2, NANOG), and upregulation of Wnt and Nodal signaling (LEF1, NODAL) and mesendodermal markers (FOXA2, TBXT) was detected, compared to mTeSR1 controls. This was confirmed by immuno-stains for FOXA2 and TBXT. On day 3, we detected a significant increase in NOTO mRNA levels in all donor lines after transfection compared to untransfected cell pellets. 3D-pellets of all donor lines showed glycosaminoglycan (GAG)- and collagen type II-rich areas after dNCM- but not TGF-β1-treatment on day 28. This was confirmed with the DMMB-assay, showing a significantly increased GAG content in the 3D-pellets treated with dNCM compared to TGF-β1. Next to that, TIE2+-cell derived iPSC pellets contained a significantly higher GAG content after dNCM-treatment compared to the PBM-cell derived hiPSC pellets. Immunohistochemical evaluation showed a heterogeneous cell population including cells positive for chondrogenic- (ACAN, SOX9), NPC/NC- (panKRT, T), and IVD progenitor- markers (CD24, TIE2). In conclusion, using tissue-specific TIE2+-cell derived hiPSCs combined with dNCM-treatment may allow for an improved differentiation capacity indicated by the increased deposition of GAG and collagen type II-rich matrix. However, the obtained cell population is still very heterogeneous and further transcriptome analysis could unravel whether the 3D-pellets contain cells which were successfully driven towards the notochordal-lineage and how these can be enriched based on unique NC-specific markers. Next to that, delineating which epigenetic features are retained after reprogramming of these two cell lines, could shed light on the observed differences in their differentiation capacity. These insights could be used for further optimization of iPS-NLC differentiation and allow for a more purified population of mature, healthy matrix-producing NLCs. This work was funded by Horizon 2020 (no. 825925) and the Dutch Arthritis Society (LLP22). References 1Colombier, P. et al. (2020). NOTO transcription factor directs human induced pluripotent stem cell-derived mesendoderm progenitors to a notochordal fate. Cells, 9(2), 509. 2Bach, Frances C., et al. "Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix: from bench towards bedside." Oncotarget 9.41 (2018): 26507

Differentiation of human induced pluripotent stem cells towards notochordal-like cells: the role of tissue source

INTRODUCTION: Notochordal cells (NCs) are linked to a healthy intervertebral disc (IVD), and they are considered an exciting target for cell-based therapy. However, NCs are scarcely available as they are lost early in life, and attempts at in vivoexpansion have failed because NCs lose their specific phenotype. The production of Notochordal-like cells (NLCs) from human induced pluripotent stem cells (iPSCs) is a viable alternative. However, current attempts have been challenged by the low differentiation efficiency into the NC lineage. Therefore, the aim of this study was to build on the tissue-specific epigenetic memory of hiPSCs derived from IVD progenitor cells (TIE2+-cells) to improve hiPSC differentiation towards mature, matrix-producing NLCs. METHODS: hiPSCs were generated from TIE2⁺ cells of three adult donors. As a comparison, donormatched minimally invasive peripheral blood mononuclear (PBM) cell-derived iPSCs were used. Firstly, the iPSCs were differentiated into mesendodermal progenitors by Wnt pathway activation (N2B27 medium + 3µM CHIR99021)¹. Thereafter, the cells were further driven towards the NClineage by transfection with synthetic NOTO mRNA¹ and further matured using a 3D pellet culture in discogenic medium containing 10ng/mL TGF-β1. Read-out parameters included cell morphology, gene and protein expression and matrix deposition. RESULTS: Both TIE2⁺ and PBM cell-derived hiPSC showed successful differentiation towards mesendodermal progenitor cells following Wnt activation on day 2, indicated by the cells moving out of the colonies after CHIR stimulation. Accordingly, a decreased gene expression of pluripotency markers (OCT4, SOX2, NANOG), and upregulation of Wnt-target genes (LEF1, NODAL) and mesendodermal markers (TBXT, FOXA2, TBX6) was observed compared to mTESR1 controls. This was confirmed by immuno-stains for FOXA2 and TBXT. At day 3, we confirmed a 9-fold increase in NOTO mRNA levels after transfection in all donor lines. At day 28, the appearance of vacuolated NLCs was observed in both TIE2⁺ and PBM cell-derived pellet cultures confirming successful commitment towards the NC-lineage. Interestingly, while DMMB-assay detected GAG deposition in both lines, a significant increase in GAG content was seen in the TIE2⁺ cell-derived pellets. DISCUSSION & CONCLUSIONS: Tissue-specific TIE2⁺ cell-derived iPSCs may allow for an improved iPSNLC differentiation efficiency, indicated by the increased potency for deposition of GAG-rich matrix. Detailed analysis of the phenotypic markers and matrix deposited at the end of the 28 day maturation is ongoing to further document the phenotype of these iPS-NLCs. Delineating which epigenetic features are retained after reprogramming of these two cell lines, could shed light on the differences in their differentiation capacity. REFERENCES: ¹Colombier et al., 202

Hyperosmolar expansion medium improves nucleus pulposus cell phenotype

Background: Repopulating the degenerated intervertebral disc (IVD) with tissue-specific nucleus pulposus cells (NPCs) has already been shown to promote regeneration in various species. Yet the applicability of NPCs as cell-based therapy has been hampered by the low cell numbers that can be extracted from donor IVDs and their potentially limited regenerative capacity due to their degenerated phenotype. To optimize the expansion conditions, we investigated the effects of increasing culture medium osmolarity during expansion on the phenotype of dog NPCs and their ability to produce a healthy extracellular matrix (ECM) in a 3D culture model. Methods: Dog NPCs were expanded in expansion medium with a standard osmolarity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxic conditions. Following expansion, NPCs were cultured in a 3D culture model in chondrogenic culture medium with a standard osmolarity. Read-out parameters included cell proliferaton rate, morphology, phenotype and healthy ECM production. Results: Increasing the expansion medium osmolarity from 300 to 500 mOsm/L resulted in NPCs with a more rounded morphology and a lower cell proliferation rate accompanied by the expression of several healthy NPC and progenitor markers at gene ( KRT18, ACAN, COL2, CD73, CD90) and protein (ACAN, PAX1, CD24, TEK, CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of their phenotypic markers and produce healthy ECM during 3D culture independent of the oxygen level used during expansion. Conclusions: Altogether, our findings show that increasing medium osmolarity during expansion results in an NPC population with improved phenotype, which could enhance the potential of cell-based therapies for IVD regeneration

Hyperosmolar expansion medium improves nucleus pulposus cell phenotype

Background:Repopulating the degenerated intervertebral disc (IVD) with tissue-spe-cific nucleus pulposus cells (NPCs) has already been shown to promote regenerationin various species. Yet the applicability of NPCs as cell-based therapy has been ham-pered by the low cell numbers that can be extracted from donor IVDs and theirpotentially limited regenerative capacity due to their degenerated phenotype. Tooptimize the expansion conditions, we investigated the effects of increasing culturemedium osmolarity during expansion on the phenotype of dog NPCs and their abilityto produce a healthy extracellular matrix (ECM) in a 3D culture model.Methods:Dog NPCs were expanded in expansion medium with a standard osmolar-ity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxicconditions. Following expansion, NPCs were cultured in a 3D culture model in chon-drogenic culture medium with a standard osmolarity. Read-out parameters includedcell proliferaton rate, morphology, phenotype and healthy ECM production.Results:Increasing the expansion medium osmolarity from 300 to 500 mOsm/Lresulted in NPCs with a more rounded morphology and a lower cell proliferation rateaccompanied by the expression of several healthy NPC and progenitor markers atgene (KRT18, ACAN, COL2, CD73, CD90) and protein (ACAN, PAX1, CD24, TEK,CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of theirphenotypic markers and produce healthy ECM during 3D culture independent of theoxygen level used during expansion. Conclusions:Altogether, our findings show that increasing medium osmolarity duringexpansion results in an NPC population with improved phenotype, which couldenhance the potential of cell-based therapies for IVD regeneration

NOTOCHORDAL CELL-DERIVED MATRIX INHIBITS MAPK SIGNALING IN THE DEGENERATIVE DISC ENVIRONMENT

Chronic low back pain is often caused by intervertebral disc (IVD) degeneration. Preceding this degenerative process, the main cellular phenotype in the nucleus pulposus shifts from notochordal cells (NCs) to nucleus pulposus cells (NPCs). In previous studies, porcine NC-derived matrix (NCM), containing NC-secreted factors, induced matrix anabolic effects and inhibited pro-inflammatory mediators in NPCs in vitro and in degenerated canine IVDs in vivo. As the underlying mechanisms remained elusive, this study aimed to explore this with targeted gene expression and proteomic (DigiWest technology) analysis focused on inflammatory signaling pathways. After 6 hours, NCM (10 mg/mL) treatment initially stimulated pro-inflammatory mediators in canine and human NPCs in vitro and increased signaling in a chondrosarcoma derived Nuclear factor-κB reporter cell line. At protein level, NCM mainly induced changes in the Mitogen-activated protein kinase (MAPK) pathway after 72 hours. Expression of inhibitory Dual-specificity phosphatase (DUSP) proteins was increased in NCM-treated NPCs, whereas the expression of the three main pillars of the MAPK pathway (extracellular signal-regulated kinase (ERK)/cJun N-terminal kinase (JNK)/protein kinase C (PKC)) was inhibited. In follow-up validation, in vivo degenerated canine IVDs treated with an intradiscal NCM injection demonstrated increased DUSP5 and healthy nucleus pulposus marker (cytokeratin 19, Paired box 1 (PAX1), Forkhead Box F1 (FOXF1)) immunopositivity after 6 months of treatment. Altogether, NCM initially stimulated pro-inflammatory mediators in vitro, but thereafter exerts its prolonged effects by inhibiting the MAPK pathway. These findings provide insights in the underlying mechanisms involved in the instructive capacity of this naturally-derived biomaterial with the potential to serve as a cell-free NC-based therapy to treat intervertebral disc degeneration

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

BACKGROUND: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. METHODS: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. RESULTS: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. CONCLUSIONS: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Background In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab‐to‐lab variability jeopardizes the much‐needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re‐differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re‐differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re‐differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species‐specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross‐lab comparisons on NP cells worldwide

Hyperosmolar expansion medium improves nucleus pulposus cell phenotype

Background: Repopulating the degenerated intervertebral disc (IVD) with tissue-specific nucleus pulposus cells (NPCs) has already been shown to promote regeneration in various species. Yet the applicability of NPCs as cell-based therapy has been hampered by the low cell numbers that can be extracted from donor IVDs and their potentially limited regenerative capacity due to their degenerated phenotype. To optimize the expansion conditions, we investigated the effects of increasing culture medium osmolarity during expansion on the phenotype of dog NPCs and their ability to produce a healthy extracellular matrix (ECM) in a 3D culture model. Methods: Dog NPCs were expanded in expansion medium with a standard osmolarity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxic conditions. Following expansion, NPCs were cultured in a 3D culture model in chondrogenic culture medium with a standard osmolarity. Read-out parameters included cell proliferaton rate, morphology, phenotype and healthy ECM production. Results: Increasing the expansion medium osmolarity from 300 to 500 mOsm/L resulted in NPCs with a more rounded morphology and a lower cell proliferation rate accompanied by the expression of several healthy NPC and progenitor markers at gene ( KRT18, ACAN, COL2, CD73, CD90) and protein (ACAN, PAX1, CD24, TEK, CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of their phenotypic markers and produce healthy ECM during 3D culture independent of the oxygen level used during expansion. Conclusions: Altogether, our findings show that increasing medium osmolarity during expansion results in an NPC population with improved phenotype, which could enhance the potential of cell-based therapies for IVD regeneration

Notochordal Cell-Derived Matrix Inhibits Mapk Signaling In The Degenerative Disc Environment

Chronic low back pain is often caused by intervertebral disc (IVD) degeneration. Preceding this degenerative process, the main cellular phenotype in the nucleus pulposus shifts from notochordal cells (NCs) to nucleus pulposus cells (NPCs). In previous studies, porcine NC-derived matrix (NCM), containing NC-secreted factors, induced matrix anabolic effects and inhibited pro-inflammatory mediators in NPCs in vitro and in degenerated canine IVDs in vivo. As the underlying mechanisms remained elusive, this study aimed to explore this with targeted gene expression and proteomic (DigiWest technology) analysis focused on inflammatory signaling pathways. After 6 hours, NCM (10 mg/mL) treatment initially stimulated pro-inflammatory mediators in canine and human NPCs in vitro and increased signaling in a chondrosarcoma derived Nuclear factor-?B reporter cell line. At protein level, NCM mainly induced changes in the Mitogen-activated protein kinase (MAPK) pathway after 72 hours. Expression of inhibitory Dual-specificity phosphatase (DUSP) proteins was increased in NCM-treated NPCs, whereas the expression of the three main pillars of the MAPK pathway (extracellular signal-regulated kinase (ERK)/cJun N-terminal kinase (JNK)/protein kinase C (PKC)) was inhibited. In follow-up validation, in vivo degenerated canine IVDs treated with an intradiscal NCM injection demonstrated increased DUSP5 and healthy nucleus pulposus marker (cytokeratin 19, Paired box 1 (PAX1), Forkhead Box F1 (FOXF1)) immunopositivity after 6 months of treatment. Altogether, NCM initially stimulated pro-inflammatory mediators in vitro, but thereafter exerts its prolonged effects by inhibiting the MAPK pathway. These findings provide insights in the underlying mechanisms involved in the instructive capacity of this naturally-derived biomaterial with the potential to serve as a cell-free NC-based therapy to treat intervertebral disc degeneration