Immune Privilege of Primary Fetal Progenitor Cells: Optimized Safety Profile of Consistent Cell Sources for Cutaneous and Musculoskeletal Regenerative Medicine

of Progenitor Cells: Optimized Safety Profile of Consistent Cell Sources Cutaneous Regenerative Abstract Numerous heterogenous biological tissues and cell types may be valorized and processed for therapeutic material generation and product development in regenerative medicine applications. Autologous, allogenic or xenogenic cell therapies may generally be considered for transplantation, whereas the immunogenicity of the implanted final products is key in determining treatment parameters. Banked primary fetal progenitor cells constitute highly interesting candidates for allogenic cell therapy approaches, as among numerous other technical advantages, such cell types are devoid of the propensity towards recipient immune response eliciting. Selection of specific gestational ages for fetal organ donation procurement enables the eventual obtention of cultured progeny cell populations consistently retaining specific subsets of MHC antigens, allowing for evasion or modulation of recipient immune systems, as attested by transplantation of fetal progenitor cells in allogenic and xenogenic frameworks in Switzerland. The present study briefly summarizes the molecular, preclinical and clinical evidence supporting the immune-privileged status of primary fetal progenitor cells and the contribution thereof to the high therapeutic value of such cellular substrates for regenerative medicine applications.


Introduction
Novel approaches of cell therapy and tissue engineering have recently contributed to widespread combined therapeutic development efforts towards optimal restoration and optimization of tissue or organ functions [1][2][3][4]. Multifactorial considerations and extensive planning are necessary in view of selecting optimal therapeutic cell sources for product development and clinical translation of cell therapies or tissue engineering solutions.
Technological simplicity and robustness in the obtention of the rapeutic biological materials enables the sustainable manufacture of innovative autologous, allogenic or xenogenic regenerative medicine substrates [5][6][7][8][9]. However and most importantly on the clinical side, considerations about immunogenicity of bioprocessed Copy@ Anthony S de Buys Roessingh and implanted materials should arise very early in the development phases, as the elicited effects after tansplantation may complexify patient management in case of recognition and tentative rejection by recipient immune systems [6,8].

Primary fetal progenitor cells have been demonstrated
to optimally adapt to highly robust and extensive biobanking frameworks, enabling establishment of large homogenous multitiered cryopreserved cell banks (Parental, Master and Working Cell Banks) destined for Good Manufacturing Practices (GMP) production of clinical-grade Advanced Therapy Medicinal Products (ATMP) or medical devices [10][11][12][13][14][15]. Such cell types, which may be traceably isolated under stringent Transplantation Program frameworks following voluntary pregnancy terminations, fulfill numerous technical requirements of industrial-scale biotechnological processing [8][9][10][11][12]. Fetal progenitors have been shown to be stable, consistent, safe and present therapeutic potentials of utmost interest, as they have been observed to stimulate repair or regeneration of various cutaneous or musculoskeletal injuries such as ulcers, deep burns or volumetric soft-tissue losses by putatively exerting paracrine stimulation of wounded tissues [6,7,9,14,[16][17][18][19]. Absence of tumorigenicity, good cyto or biocompatibility and on-demand availability, rapid proliferation, pre-terminal differentiation and high resistance to oxidative stress are strong advantages of these cell types, yet the paramount clinical advantages conferred by the specific absence of observable immunogenicity allow for pragmatic consideration of such cellular substrates for simple and effective management of acute and degenerative cutaneous and musculoskeletal affections [12,15]. Transplantation Program and types of preclinical or clinical applications documented to date evidencing absence of cell therapy recipient immune system activation. A) Different tissue biopsies are simultaneously provided from one organ donation after micro-dissection. B) Tissues are further dissected into fragments allowing for enzymatic or mechanical cell culture initiation. C) Culture-expanded cell populations are cryopreserved to form homogenous and extensive cell banks. D) Cell vials are thawed and contents may be directly used or further processed in oder to produce cell therapies or cell-based therapeutic products. E) Schematic illustrations of the different patient/animal models which have been treated under the different Swiss Fetal Progenitor Cell Transplantation Programs with primary fetal progenitor cells without presenting signs of immune recognition or rejection to date. All species were treated with human fetal progenitor cells (allogenic treatment, human to human or xenogenic treatment, human to animal) except horses, which were treated with allogenic equine primary progenitor cell types. See Table 1 for specific references. Table 1: Overview of the different types of cell transplantation (allogenic and xenogenic) using cultured primary fetal progenitor cells after primary cell type establishement and banking in Switzerland. Of all the considered preclinical and clinical applications listed herein, none provided evidence of recipient immune system activation by transplanted therapeutic cellular products.

Immune Privilege of Fetal Progenitor Cell Types
Fetal progenitor cell types and mesenchymal stem cells (MSC) display similar specific immunological profiles, as they may be considered to be immune-privileged or classified as possessing immunomodulatory capabilities [13]. It is clear that the gestational age of the fetus at the time of pregnancy termination and organ donation plays a paramount role in the molecular characteristics of derived progeny cultured cells. Indeed, major histo-compatibility complex (MHC) antigen expression during fetal development is organ and gestational age-specific [26]. Fetal progenitor cells are considered pre-immunocompetent and may fail in eliciting immunological responses due to an observed lack of post-thymic T lymphocytes in the first 13 gestational weeks [5,27]. It is well accepted that T lymphocytes are heavily implicated in allograft rejection mechanisms, mainly through differential human leucocyte antigen (HLA) recognition, while expression of surface MHC class I proteins typically triggers T CD8-mediated immune reactions [13].
In the particular and comparative case of stem cell studies however, it was shown that MSCs expressing MHC class I antigens could be grafted without eliciting an immunological response. Such cell types have been also shown to have proliferation inhibition capabilities towards T lymphocytes cultured in vitro [28]. Transplantation in primate models has yielded some evidence of low immunogenicity, despite mismatches in MHC panel expression [29,30]. Additionally, neonatal cell types were shown to display similar MHC profiles while presenting acceptable safety (e.g. neonatal foreskin keratinocytes) [31]. By extrapolation, it is probable that primary fetal progenitor cells possess capabilities and behaviors similar to those of MSCs in their interactions with recipient immune system effectors, albeit being different from MSCs in terms of potency [13]. Indeed, primary fetal progenitor cell types generally lack MHC class II proteins (HLA-DP, DQ, DR) and exhibit relatively low levels of MHC class I (HLA-A, B, C) counterparts, therefore closely resembling marker panels yielded by MSCs or neonatal foreskin keratinocytes [13,28,31,32].
Such observed tolerance is highly beneficial for the use of allogenic primary progenitors, as adjuvant immunosuppressive treatments are not necessary after transplantation to immune-competent patients.
Specific fetal tissues were shown to selectively express HLA-G antigens, known mediators of tolerogenic effects [33,34].

Immune-modulation and inhibition of lymphoproliferation by
fetal progenitor cells is also attributed to specific expression of indoleamine 2,3-dioxygenase (IDO) [28,30,35]. Fetal cells definitely possess immune system modulation capabilities, as attested during the gestational development, where an immunologic equilibrium exists between the mother and the embryo or fetus. Indeed, absence of immune reaction triggering despite in utero recognition of paternal HLA-C markers and modulatory effects of HLA-G antigens on lymphocytic activity characterize the particular immune status of fetal tissues during gestation and thus that of derived fetal progenitor cells [33,[36][37][38]. Futhermore, it was also shown that fetal cells persist in the mother's tissues for extended periods after delivery and may contribute to healing of maternal injuries [39].
When considering implantation in an immunocompetent recipient, it is possible that multimodal mechanisms are deployed by fetal progenitor cells, leading to localized redefinition of immune microenvironments at the site of delivery, allowing for early inflammation control and stimulation of physiological repair processes [11].

Preclinical and Clinical Immune Tolerance of Fetal Progenitor Cells
Extensive preclinical experience has been gathered around the use of primary fetal progenitor cells in Switzerland and in subsequent international collaborations [15].  [6,8,11,13,14,[21][22][23][24]. Implantation of human progenitor cells in various xenogenic frameworks (murine, rat, caprine, porcine and rabbit models) have not yielded evidence of immune system activation in immunocompetent animals [13,15,18,19,23,24]. Extensive did not lead to immune system activation [18,19]. Moreover, the observed clinical effects after fetal progenitor cell applications rather comprised potent anti-inflammatory components and pain reduction, suggesting an active process or interplay with the recipient organism, rather than a simple passive immune system evasion [16,19]. In addition, extended studies and hindsight also indicate an absence of tumorigenicity of primary fetal progenitor cells, which might be due to the inherent relatively high cell stability during processing [15].