Human muscular fetal cells: a potential cell source for muscular therapies

Myoblast transfer therapy has been extensively studied for a wide range of clinical applications, such as tissue engineering for muscular loss, cardiac surgery or Duchenne Muscular Dystrophy treatment. However, this approach has been hindered by numerous limitations, including early myoblast death after injection and specific immune response after transplantation with allogenic cells. Different cell sources have been analyzed to overcome some of these limitations. The object of our study was to investigate the growth potential, characterization and integration in vivo of human primary fetal skeletal muscle cells. These data together show the potential for the creation of a cell bank to be used as a cell source for muscle cell therapy and tissue engineering. For this purpose, we developed primary muscular cell cultures from biopsies of human male thigh muscle from a 16-week-old fetus and from donors of 13 and 30years old. We show that fetal myogenic cells can be successfully isolated and expanded in vitro from human fetal muscle biopsies, and that fetal cells have higher growth capacities when compared to young and adult cells. We confirm lineage specificity by comparing fetal muscle cells to fetal skin and bone cells in vitro by immunohistochemistry with desmin and 5.1H11 antibodies. For the feasibility of the cell bank, we ensured that fetal muscle cells retained intrinsic characteristics after 5years cryopreservation. Finally, human fetal muscle cells marked with PKH26 were injected in normal C57BL/6 mice and were found to be present up to 4days. In conclusion we estimate that a human fetal skeletal muscle cell bank can be created for potential muscle cell therapy and tissue engineerin

Epiphyseal chondro-progenitors provide a stable cell source for cartilage cell therapy

Articular cartilage regeneration poses particularly tough challenges for implementing cell-­‐based therapies. Many cell types have been investigated looking for a balanced combination of responsiveness and stability, yet techniques are still far from defining a gold standard. The work presented focuses on the reliable expansion and characterization of a clinical-­‐grade human epiphyseal chondro-­‐progenitor (ECP) cell bank from a single tissue donation. A parental human ECP cell bank was established which provides the seed material for master and working cell banks. ECPs were investigated at both low and high cumulative population doublings looking at morphology, monolayer expansion kinetics, resistance to cryogenic shock, colony forming efficiency and cell surface markers. Three dimensional micro-­‐pellet assays were used to determine spontaneous extracellular matrix deposition at varying population doublings and monolayer 2D differentiation studies were undertaken to assess the propensity for commitment into other lineages and their stability. ECPs exhibited remarkable homogeneity in expansion with a steady proliferative potential averaging 3 population doublings over eight days. Surface marker analysis revealed no detectable contaminating subpopulations or population enrichment during prolonged culture periods. Despite a slight reduction in Sox9 expression levels at higher population doublings in monolayer, nuclear localization was equivalent both in monolayer and in micro-­‐pellet format. Equally, ECPs were capable of depositing glycosaminoglycans, producing aggrecan, collagen I and collagen II in 3D pellets both at after low and high population doublings indicating a stable spontaneous chondrogenic potential. Osteogenic induction was differentially restricted in low and high population doublings as observed by Von Kossa staining of calcified matrix, with a notable collagen X, MMP13 and ADAMTS5 down-­‐regulation. Rare adipogenic induction was seen as evidenced by cytoplasmic lipid accumulation detectable by Oil Red O staining. These findings highlight the reliability, stability and responsiveness of ECPs over prolonged culture, making them ideal candidates in defining novel strategies for cartilage regeneration

Biologicals and Fetal Cell Therapy for Wound and Scar Management

Few biopharmaceutical preparations developed from biologicals are available for tissue regeneration and scar management. When developing biological treatments with cellular therapy, selection of cell types and establishment of consistent cell banks are crucial steps in whole-cell bioprocessing. Various cell types have been used in treatment of wounds to reduce scar to date including autolog and allogenic skin cells, platelets, placenta, and amniotic extracts. Experience with fetal cells show that they may provide an interesting cell choice due to facility of outscaling and known properties for wound healing without scar. Differential gene profiling has helped to point to potential indicators of repair which include cell adhesion, extracellular matrix, cytokines, growth factors, and development. Safety has been evidenced in Phase I and II clinical fetal cell use for burn and wound treatments with different cell delivery systems. We present herein that fetal cells present technical and therapeutic advantages compared to other cell types for effective cell-based therapy for wound and scar management

Wound healing gene-family expression differences between fetal and foreskin cells used for bioengineered skin substitutes

For tissue engineering, several cell types and tissues have been proposed as starting material. Allogenic skin products available for therapeutic usage are mostly developed with cell culture and with foreskin tissue of young individuals. Fetal skin cells offer a valuable solution for effective and safe tissue engineering for wounds due to their rapid growth and simple cell culture. By selecting families of genes that have been reported to be impli- cated in wound repair and particularly for scarless fetal wound healing including transforming growth factor-beta (TGF-b) superfamily, extracellular matrix, and nerve/angiogenesis growth factors, we have analyzed differences in their expression between fetal skin and foreskin cells, and the same passages. Of the five TGF-b superfamily genes analyzed by real-time reverse transcription–polymerase chain reaction, three were found to be signifi- cantly different with sixfold up-regulated for TGF-b2, and 3.8-fold for BMP- 6 in fetal cells, whereas GDF-10 was 11.8-fold down-regulated. For nerve growth factors, midkine was 36-fold down-regulated in fetal cells, and pleiotrophin was 4.76-fold up-regulated. We propose that fetal cells present technical and therapeutic advantages compared to foreskin cells for effective cell-based therapy for wound management, and overall differences in gene expression could contribute to the degree of efficiency seen in clinical use with these cells

Industrial Development of Standardized Fetal Progenitor Cell Therapy for Tendon Regenerative Medicine: Preliminary Safety in Xenogeneic Transplantation.

Tendon defects require multimodal therapeutic management over extensive periods and incur high collateral burden with frequent functional losses. Specific cell therapies have recently been developed in parallel to surgical techniques for managing acute and degenerative tendon tissue affections, to optimally stimulate resurgence of structure and function. Cultured primary human fetal progenitor tenocytes (hFPT) have been preliminarily considered for allogeneic homologous cell therapies, and have been characterized as stable, consistent, and sustainable cell sources in vitro. Herein, optimized therapeutic cell sourcing from a single organ donation, industrial transposition of multi-tiered progenitor cell banking, and preliminary preclinical safety of an established hFPT cell source (i.e., FE002-Ten cell type) were investigated. Results underlined high robustness of FE002-Ten hFPTs and suitability for sustainable manufacturing upscaling within optimized biobanking workflows. Absence of toxicity or tumorigenicity of hFPTs was demonstrated in ovo and in vitro, respectively. Furthermore, a 6-week pilot good laboratory practice (GLP) safety study using a rabbit patellar tendon partial-thickness defect model preliminarily confirmed preclinical safety of hFPT-based standardized transplants, wherein no immune reactions, product rejection, or tumour formation were observed. Such results strengthen the rationale of the multimodal Swiss fetal progenitor cell transplantation program and prompt further investigation around such cell sources in preclinical and clinical settings for musculoskeletal regenerative medicine

Development of Standardized Fetal Progenitor Cell Therapy for Cartilage Regenerative Medicine: Industrial Transposition and Preliminary Safety in Xenogeneic Transplantation

Diverse cell therapy approaches constitute prime developmental prospects for managing acute or degenerative cartilaginous tissue affections, synergistically complementing specific surgical solutions. Bone marrow stimulation (i.e., microfracture) remains a standard technique for cartilage repair promotion, despite incurring the adverse generation of fibrocartilagenous scar tissue, while matrix-induced autologous chondrocyte implantation (MACI) and alternative autologous cell-based approaches may partly circumvent this effect. Autologous chondrocytes remain standard cell sources, yet arrays of alternative therapeutic biologicals present great potential for regenerative medicine. Cultured human epiphyseal chondro-progenitors (hECP) were proposed as sustainable, safe, and stable candidates for chaperoning cartilage repair or regeneration. This study describes the development and industrial transposition of hECP multi-tiered cell banking following a single organ donation, as well as preliminary preclinical hECP safety. Optimized cell banking workflows were proposed, potentially generating millions of safe and sustainable therapeutic products. Furthermore, clinical hECP doses were characterized as non-toxic in a standardized chorioallantoic membrane model. Lastly, a MACI-like protocol, including hECPs, was applied in a three-month GLP pilot safety evaluation in a caprine model of full-thickness articular cartilage defect. The safety of hECP transplantation was highlighted in xenogeneic settings, along with confirmed needs for optimal cell delivery vehicles and implantation techniques favoring effective cartilage repair or regeneration

Industrial Development of Standardized Fetal Progenitor Cell Therapy for Tendon Regenerative Medicine: Preliminary Safety in Xenogeneic Transplantation

Tendon defects require multimodal therapeutic management over extensive periods and incur high collateral burden with frequent functional losses. Specific cell therapies have recently been developed in parallel to surgical techniques for managing acute and degenerative tendon tissue affections, to optimally stimulate resurgence of structure and function. Cultured primary human fetal progenitor tenocytes (hFPT) have been preliminarily considered for allogeneic homologous cell therapies, and have been characterized as stable, consistent, and sustainable cell sources in vitro. Herein, optimized therapeutic cell sourcing from a single organ donation, industrial transposition of multi-tiered progenitor cell banking, and preliminary preclinical safety of an established hFPT cell source (i.e., FE002-Ten cell type) were investigated. Results underlined high robustness of FE002-Ten hFPTs and suitability for sustainable manufacturing upscaling within optimized biobanking workflows. Absence of toxicity or tumorigenicity of hFPTs was demonstrated in ovo and in vitro, respectively. Furthermore, a 6-week pilot good laboratory practice (GLP) safety study using a rabbit patellar tendon partial-thickness defect model preliminarily confirmed preclinical safety of hFPT-based standardized transplants, wherein no immune reactions, product rejection, or tumour formation were observed. Such results strengthen the rationale of the multimodal Swiss fetal progenitor cell transplantation program and prompt further investigation around such cell sources in preclinical and clinical settings for musculoskeletal regenerative medicine

Wassergefilterte Infrarot-A-Strahlung (wIRA) ist nicht an der Zelldegeneration menschlicher Haut beteiligt

Background: Excessive exposure to solar ultraviolet radiation is involved in the complex biologic process of cutaneous aging. Wavelengths in the ultraviolet-A and -B range (UV-A and UV-B) have been shown to be responsible for the induction of proteases, e. g. the collagenase matrix metalloproteinase 1 (MMP-1), which are related to cell aging. As devices emitting longer wavelengths are widely used in therapeutic and cosmetic interventions and as the induction of MMP-1 by water-filtered infrared-A (wIRA) had been discussed, it was of interest to assess effects of wIRA on the cellular and molecular level known to be possibly involved in cutaneous degeneration. Objectives: Investigation of the biological implications of widely used water-filtered infrared-A (wIRA) radiators for clinical use on human skin fibroblasts assessed by MMP-1 gene expression (MMP-1 messenger ribonucleic acid (mRNA) expression). Methods: Human skin fibroblasts were irradiated with approximately 88% wIRA (780-1400 nm) and 12% red light (RL, 665-780 nm) with 380 mW/cm² wIRA(+RL) (333 mW/cm² wIRA) on the one hand and for comparison with UV-A (330-400 nm, mainly UV-A1) and a small amount of blue light (BL, 400-450 nm) with 28 mW/cm² UV-A(+BL) on the other hand. Survival curves were established by colony forming ability after single exposures between 15 minutes and 8 hours to wIRA(+RL) (340-10880 J/cm² wIRA(+RL), 300-9600 J/cm² wIRA) or 15-45 minutes to UV-A(+BL) (25-75 J/cm² UV-A(+BL)). Both conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and quantitative real-time RT-PCR techniques were used to determine the induction of MMP-1 mRNA at two physiologic temperatures for skin fibroblasts (30°C and 37°C) in single exposure regimens (15-60 minutes wIRA(+RL), 340-1360 J/cm² wIRA(+RL), 300-1200 J/cm² wIRA; 30 minutes UV-A(+BL), 50 J/cm² UV-A(+BL)) and in addition at 30°C in a repeated exposure protocol (up to 10 times 15 minutes wIRA(+RL) with 340 J/cm² wIRA(+RL), 300 J/cm² wIRA at each time). Results: Single exposure of cultured human dermal fibroblasts to UV-A(+BL) radiation yielded a very high increase in MMP-1 mRNA expression (11 ±1 fold expression for RT-PCR and 76 ±2 fold expression for real-time RT-PCR both at 30°C, 75 ±1 fold expression for real-time RT-PCR at 37°C) and a dose-dependent decrease in cell survival. In contrast, wIRA(+RL) did not produce cell death and did not induce a systematic increase in MMP-1 mRNA expression (less than twofold expression, within the laboratory range of fluctuation) detectable with the sensitive methods applied. Additionally, repeated exposure of human skin fibroblasts to wIRA(+RL) did not induce MMP-1 mRNA expression systematically (less than twofold expression by up to 10 consecutive wIRA(+RL) exposures and analysis with real-time RT-PCR). Conclusions: wIRA(+RL) even at the investigated disproportionally high irradiances does not induce cell death or a systematic increase of MMP-1 mRNA expression, both of which can be easily induced by UV-A radiation. Furthermore, these results support previous findings of in vivo investigations on collagenase induction by UV-A but not wIRA and show that infrared-A with appropriate irradiances does not seem to be involved in MMP-1 mediated photoaging of the skin. As suggested by previously published studies wIRA could even be implicated in a protective manner.Hintergrund: Eine übermäßige Exposition gegenüber Ultraviolettstrahlung der Sonne ist mit dem komplexen biologischen Prozess der Hautalterung verbunden. Für Wellenlängen im Ultraviolett A und B (UVA und UVB) wurde gezeigt, dass sie für die mit der Zellalterung verbundene Protease-Induktion, z. B. der Kollagenase Matrix-Metalloproteinase 1 (MMP-1), mitverantwortlich sind. Da längere Wellenlängen häufig für therapeutische und kosmetische Zwecke verwendet werden und die Induktion von MMP-1 durch wassergefiltertes Infrarot A (wIRA) diskutiert worden war, war es von Interesse, Effekte von wIRA, die zur Hautdegeneration führen könnten, auf zellulärem und molekularem Niveau zu untersuchen. Ziele: Untersuchung der biologischen Wirkungen von klinisch häufig gebrauchten wassergefilterten Infrarot-A-Strahlern auf menschliche Hautfibroblasten anhand der MMP-1-Gen-Expression (Expression der MMP-1-Messenger-Ribonukleinsäure (mRNA)). Methoden: Menschliche Hautfibroblasten wurden einerseits etwa 88% wIRA (780-1400 nm) und etwa 12% rotem Licht (RL, 665-780 nm) mit 380 mW/cm² wIRA(+RL) (333 mW/cm² wIRA) und andererseits zum Vergleich UVA-Strahlung (330-400 nm, vorwiegend UVA1) und einem kleinen Anteil von blauem Licht (BL, 400-450 nm) mit 28 mW/cm² UVA(+BL) ausgesetzt. Es wurden Überlebenskurven anhand der Koloniebildungsfähigkeit nach Einzelbestrahlungen zwischen 15 Minuten und 8 Stunden mit wIRA(+RL) (340-10880 J/cm² wIRA(+RL), 300-9600 J/cm² wIRA) oder 15-45 Minuten mit UVA(+BL) (25-75 J/cm² UVA(+BL)) erstellt. Sowohl die konventionelle Reverse-Transkriptase-Polymerase-Kettenreaktion (RT-PCR) als auch die quantitative Echtzeit-RT-PCR wurden angewandt, um die Induktion von MMP-1-mRNA bei zwei physiologischen Temperaturen für Hautfibroblasten (30°C und 37°C) nach Einzelexposition (15-60 Minuten wIRA(+RL), 340-1360 J/cm² wIRA(+RL), 300-1200 J/cm² wIRA; 30 Minuten UVA(+BL), 50 J/cm² UVA(+BL)) und zusätzlich bei 30°C nach wiederholter Exposition (bis zu zehnmal 15 Minuten wIRA(+RL) mit bei jedem Mal 340 J/cm² wIRA(+RL), 300 J/cm² wIRA) zu bestimmen. Ergebnisse: Nach einer einzelnen UVA(+BL)-Exposition von kultivierten menschlichen dermalen Fibroblasten zeigte sich eine sehr starke Zunahme der MMP-1-mRNA-Expression (11 ±1 fache Expression für RT-PCR und 76 ±2 fache Expression für Echtzeit-RT-PCR jeweils bei 30°C, 75 ±1 fache Expression für Echtzeit-RT-PCR bei 37°C) sowie eine dosisabhängige Minderung des Zellüberlebens. Im Gegensatz hierzu rief wIRA(+RL) kein Zellsterben und keine mit den angewendeten sensitiven Methoden erkennbare systematische Induktion der MMP-1-mRNA-Expression hervor (weniger als zweifache Expression, innerhalb der methodischen Schwankungsbreite). Auch bei wiederholter wIRA(+RL)-Exposition von menschlichen Hautfibroblasten wurde MMP-1-mRNA nicht systematisch induziert (weniger als zweifache Expression bei bis zu 10 aufeinanderfolgenden wIRA(+RL)-Expositionen und Analyse mit Echtzeit-RT-PCR). Folgerungen: wIRA(+RL) induziert selbst unter den untersuchten unphysiologisch hohen Bestrahlungsstärken im Gegensatz zu UVA-Strahlung weder den Zelltod noch eine systematisch verstärkte Expression von MMP-1-mRNA. Diese Ergebnisse unterstützen Resultate früherer in vivo-Untersuchungen zur Kollagenase-Induktion durch UVA, aber nicht durch wIRA, und zeigen, dass Infrarot A bei adäquaten Bestrahlungsstärken nicht in die MMP-1-induzierte Lichtalterung der Haut involviert zu sein scheint. Wie in früher veröffentlichten Studien nahegelegt, könnte wIRA sogar eher mit einer Schutzfunktion verbunden sein