Pre-conditioning with IFN-γ and hypoxia enhances the angiogenic potential of iPSC-Derived MSC secretome

Induced pluripotent stem cell (iPSC) derived mesenchymal stem cells (iMSCs) represent a promising source of progenitor cells for approaches in the field of bone regeneration. Bone formation is a multi-step process in which osteogenesis and angiogenesis are both involved. Many reports show that the secretome of mesenchymal stromal stem cells (MSCs) influences the microenvironment upon injury, promoting cytoprotection, angiogenesis, and tissue repair of the damaged area. However, the effects of iPSC-derived MSCs secretome on angiogenesis have seldom been investigated. In the present study, the angiogenic properties of IFN-γ pre-conditioned iMSC secretomes were analyzed. We detected a higher expression of the pro-angiogenic genes and proteins of iMSCs and their secretome under IFN-γ and hypoxic stimulation (IFN-H). Tube formation and wound healing assays revealed a higher angiogenic potential of HUVECs in the presence of IFN-γ conditioned iMSC secretome. Sprouting assays demonstrated that within Coll/HA scaffolds, HUVECs spheroids formed significantly more and longer sprouts in the presence of IFN-γ conditioned iMSC secretome. Through gene expression analyses, pro-angiogenic genes (FLT-1, KDR, MET, TIMP-1, HIF-1α, IL-8, and VCAM-1) in HUVECs showed a significant up-regulation and down-regulation of two anti-angiogenic genes (TIMP-4 and IGFBP-1) compared to the data obtained in the other groups. Our results demonstrate that the iMSC secretome, pre-conditioned under inflammatory and hypoxic conditions, induced the highest angiogenic properties of HUVECs. We conclude that pre-activated iMSCs enhance their efficacy and represent a suitable cell source for collagen/hydroxyapatite with angiogenic properties.S.W. was financed by the China Scholarship Council (CSC) (grant 201908080045)

Optimierung von Kieferperiostzellen für das Knochen-Tissue Engineering: Herstellung von iPS-Zellen und Analyse von Progenitor-Oberflächenmarkern

Die Knochengewebezüchtung im Labor (engl.: Bone Tissue Engineering, BTE) könnte in Zukunft autologe Knochentransplantate in der Mund-, Kiefer- und Gesichtschirurgie (MKGC) ersetzen. Für die Herstellung klinisch anwendbarer BTE- Produkte sind die Qualität und die Verfügbarkeit der dabei eingesetzten Stammzellen entscheidende Faktoren. Die Verfügbarkeit von Stammzellen ist einerseits durch die Größe der Gewebebiopsie, andererseits durch die Abnahme der Vitalität und des osteogenen Potenzials im Laufe der in vitro Kultivierung limitiert. Da für die Herstellung von BTE-Konstrukten zur Regeneration größerer Knochendefekte, z.B. nach Tumorresektionen, große Mengen osteogener Vorläuferzellen benötigt werden, sollten in der vorliegenden Arbeit induzierte pluripotente Stammzellen (iPSCs) aus Kieferperiostzellen (JPCs) generiert, und als Zellquelle für das BTE etabliert werden. iPSCs besitzen die Fähigkeit zur Selbsterneuerung und sind daher in großen Mengen verfügbar. Im Hinblick auf zukünftige klinische Anwendungen der Zellen, wurde zur Generierung von iPSCs eine besonders sichere xenogen-freie RNA-basierte Reprogrammierungstechnik entwickelt. Durch die Differenzierung von iPSCs zu MSC-ähnlichen Zellen (iMSCs) ist es möglich osteogene Vorläuferzellen herzustellen, welche analog zu normalen MSCs (mesenchymale Stamm-/Stromazellen) eingesetzt werden können. In der vorliegenden Arbeit wurden daher aus JPCs generierte iPSCs zu iMSCs differenziert, und ihre Eignung für Anwendungen im BTE anhand der osteogenen Differenzierung in vitro getestet. Neben der Verfügbarkeit von Stammzellen ist auch die Qualität des zur Verfügung stehenden Zellmaterials ein entscheidender Faktor. Die Gewinnung von Stammzellen aus Gewebebiopsien führt jedoch meistens zur Isolation von heterogenen Zellpopulationen aus verschiedenen Zelltypen, wodurch die Qualität sowie das Stammzellpotenzial der für das BTE benötigten Zellkulturen beeinträchtigt werden kann. Zur Qualitätskontrolle und zur Isolation osteogener Vorläuferzellen aus solchen Mischpopulationen werden Oberflächenmarker benötigt, mit denen sich osteogene Vorläuferzellen in heterogenen Zellpopulationen identifizieren und ggf. mithilfe von Fluorescence-activated Cell Sorting (FACS) isolieren lassen. In der vorliegenden Arbeit wurden die Oberflächenmarker MSCA-1 (Mesenchymal Stromal Cell Antigen-1) und CD146 (Melanoma Cell Adhesion Molecule (MCAM)) auf ihre Eignung für die Separation osteogener Vorläuferzellen aus der Periostzelllinie TAg58 getestet. Dabei zeigte sich, dass obwohl beide Marker in der Literatur als osteogene Marker beschrieben waren, sich nur MSCA-1 zur Identifikation und Sortierung osteogener Vorläuferzellen in der verwendeten Zelllinie als geeignet erwies. Zur Herstellung von BTE-Produkten unter GMP-gerechten Bedingungen sollte bei der Zellkultur auf xenogene Bestandteile verzichtet werden. Daher wurde im Zuge dieser Arbeit die Kultivierung von JPCs von FBS- (fötales Kälberserum) auf hPL- (humanes Plättchenlysat) supplementiertes Medium umgestellt. Dabei zeigte sich, dass sich hPL wesentlich besser für die Kultivierung von JPCs eignete und Proliferation sowie Differenzierungspotenzial deutlich verbessert wurden. Zudem konnte für die osteogene Induktion von Periostzellen auf die Zugabe von Dexamethason unter hPL-Supplementierung verzichtet werden

Jaw Periosteum-Derived Mesenchymal Stem Cells Regulate THP-1-Derived Macrophage Polarization

Mesenchymal stem cells from bone marrow have powerful immunomodulatory capabilities. The interactions between jaw periosteal cells (JPCs) and macrophages are not only relevant for the application of JPCs in regenerative medicine, but this understanding could also help treating diseases like osteonecrosis of the jaw. In previous studies, we analyzed, for the first time, immunomodulatory features of 2D- and 3D-cultured JPCs. In the present work, the effects of JPCs on the polarization state of macrophages in contact coculture were analyzed. To improve the macrophage polarization study, different concentrations of PMA (5 nM, 25 nM, and 150 nM) or different medium supplementations (10% FBS, 10% hPL and 5% hPL) were compared. Further, in order to analyze the effects of JPCs on macrophage polarization, JPCs and PMA-stimulated THP-1 cells were cocultured under LPS/IFN-γ or IL-4/IL-13 stimulatory conditions. Surface marker expression of M1 and M2 macrophages were analyzed under the different culture supplementations in order to investigate the immunomodulatory properties of JPCs. Our results showed that 5 nM PMA can conduct an effective macrophage polarization. The analyses of morphological parameters and surface marker expression showed more distinct M1/M2 phenotypes over FBS supplementation when using 5% hPL during macrophage polarization. In the coculture, immunomodulatory properties of JPCs improved significantly under 5% hPL supplementation compared to other supplementations. We concluded that, under the culture condition with 5% hPL, JPCs were able to effectively induce THP-1-derived macrophage polarization

Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization

Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells

Impact of Fluid Dynamics on the Viability and Differentiation Capacity of 3D-Cultured Jaw Periosteal Cells

Perfused bioreactor systems are considered to be a promising approach for the 3D culturing of stem cells by improving the quality of the tissue-engineered grafts in terms of better cell proliferation and deeper penetration of used scaffold materials. Our study aims to establish an optimal perfusion culture system for jaw periosteal cell (JPC)-seeded scaffolds. For this purpose, we used beta-tricalcium phosphate (β-TCP) scaffolds as a three-dimensional structure for cell growth and osteogenic differentiation. Experimental set-ups of tangential and sigmoidal fluid configurations with medium flow rates of 100 and 200 µL/min were applied within the perfusion system. Cell metabolic activities of 3D-cultured JPCs under dynamic conditions with flow rates of 100 and 200 µL/min were increased in the tendency after 1, and 3 days of culture, and were significantly increased after 5 days. Significantly higher cell densities were detected under the four perfused conditions compared to the static condition at day 5. However, cell metabolic and proliferation activity under dynamic conditions showed flow rate independency in our study. In this study, dynamic conditions increased the expression of osteogenic markers (ALPL, COL1A1, RUNX2, and OCN) compared to static conditions and the tangential configuration showed a stronger osteogenic effect than the sigmoidal flow configuration

Angiogenic Potential of VEGF Mimetic Peptides for the Biofunctionalization of Collagen/Hydroxyapatite Composites

Currently, the focus on bioinspired concepts for the development of tissue engineering constructs is increasing. For this purpose, the combination of collagen (Coll) and hydroxyapatite (HA) comes closest to the natural composition of the bone. In order to confer angiogenic properties to the scaffold material, vascular endothelial growth factor (VEGF) is frequently used. In the present study, we used a VEGF mimetic peptide (QK) and a modified QK-peptide with a poly-glutamic acid tag (E7-QK) to enhance binding to HA, and analyzed in detail binding efficiency and angiogenic properties. We detected a significantly higher binding efficiency of E7-QK peptides to hydroxyapatite particles compared to the unmodified QK-peptide. Tube formation assays revealed similar angiogenic functions of E7-QK peptide (1µM) as induced by the entire VEGF protein. Analyses of gene expression of angiogenic factors and their receptors (FLT-1, KDR, HGF, MET, IL-8, HIF-1α, MMP-1, IGFBP-1, IGFBP-2, VCAM-1, and ANGPT-1) showed higher expression levels in HUVECs cultured in the presence of 1 µM E7-QK and VEGF compared to those detected in the negative control group without any angiogenic stimuli. In contrast, the expression of the anti-angiogenic gene TIMP-1 showed lower mRNA levels in HUVECs cultured with E7-QK and VEGF. Sprouting assays with HUVEC spheroids within Coll/HA/E7-QK scaffolds showed significantly longer sprouts compared to those induced within Coll/HA/QK or Coll/HA scaffolds. Our results demonstrate a significantly better functionality of the E7-QK peptide, electrostatically bound to hydroxyapatite particles compared to that of unmodified QK peptide. We conclude that the used E7-QK peptide represents an excellently suited biomolecule for the generation of collagen/hydroxyapatite composites with angiogenic properties