# Biologicals and Fetal Cell Therapy for Wound and Scar Management

Authors: Nathalie Hirt-Burri; Albert-Adrien Ramelet; Wassim Raffoul; Anthony de Buys Roessingh; Corinne Scaletta; Dominique Pioletti; Lee Ann Applegate
Journal: ISRN Dermatology
 (2011)

---

## Abstract

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.


---

## Body

## 1. Introduction

Cell-based therapies are penetrating gently into routine medical care and especially for wound management of skin. They offer the promise of repairing and/or replacing damaged tissue and restoring lost functionality, because ideally, they provide all of the factors necessary for wound healing. Several cell types and tissues have been proposed as starting material including autologous cells, adult stem cells including those derived from bone marrow, and adipose tissue, fetal cells, embryonic stem cells, platelets, and tissues from placental and amniotic fluid. These cell types are used for biological preparations in processing vaccines and medicinal, veterinary, and tissue engineering products [1–41].  As the literature and information is vast on cell-based therapies, this paper will concentrate on fetal cells as the choice in wound and scar management. Firstly, we will define differences between stem, and mesenchymal and fetal cells, as the literature is confusing with these terminologies, followed by a short review of fetal wound healing and associated processes. Importantly, cell choice and the technical specifications to outscale, stability, safety, and delivery are the major hurdles for development of biologicals for better wound treatments and scar management. Fetal skin cells present biological, technical, and therapeutic advantages lending towards possible routine cellular-based therapy for wound and scar management. All of these aspects will be addressed in the description of how dedicated fetal skin cell banks can be developed, potential delivery systems, and cellular mechanisms of repair with gene profile differences between fetal and young skin cells to illustrate biological families implicated in wound healing. Finally, the capacity of this cell type in wound and scar management is illustrated and summarized from Phase I and II clinical safety studies in humans.


### 1.1. Cellular Sources as Therapeutic Agents: Terminology Clarification, Technical Requirements, and Cell Banking

There is some confusion between the terminology and potential of embryonic, fetal, and adult stem cells. These cells are referred to in the literature as embryonic stem cells, fetal cells, and mesenchymal stem cells, respectively. However, more frequently, all of these cell types are referred to as simply stem cells, neglecting all of the legal and technical aspects associated with each specific cell type.  To illustrate these differences, Figure1 lists the major cell sources used in developing therapeutic applications showing that some cell choices are more adaptable to cellular therapy in patients. This adaptability is highly associated with technical facility of expanding and selecting cell populations needed. Tissue choices from animal and human at all ages of development can be evaluated with advantages and disadvantages for each final cell type (Figure 1).  In legal aspects, the term “embryo” denotes the earliest stages following fertilization of an ovum by a sperm. Zygote would include early stage cleavage embryos produced by cell division up to 50–60 cell stage (each cell which is a blastomere) and the blastocyte for the 60 cell stage to the point of implantation at about 2 weeks after-fertilization. “Embryonic stem cells” (ES) are developed from preimplantation embryos from the inner-cell mass before the first 2 weeks of development. These cells are frequently obtained from extra embryos developed by “in vitro” fertilization techniques to aid couples for fertility purposes. Because these particular cells have created quite an ethical debate, other researchers have begun using “fetal embryonic cells” derived from voluntary interruption of pregnancy between 5 and 8 weeks. Cell lines are normally developed from the genital ridge of the fetus. As this tissue is considered as an organ donation in most countries, it bypasses the major problems that have been raised by embryonic stem cells. Most fetal cell research is developed from specific tissues such as skin, muscle bone, or cartilage at the latter end of the first trimester (11–14 weeks) following voluntary interruption of pregnancies. Cell lines at this stage are tissue specific, and therefore, “fetal cells” are differentiated with specific functions. Fetal cells are derived from a legal organ donation when the mother donor ensures informed consent, provided that the tissue is a donation and not paid for and that there is no change in timing or method of pregnancy interruption for the sake of research. As fetal cells are from organ donations and under transplantation medicine, they are also routinely included in the terminology as adult stem cells.Figure 1
Cellular source and development stage with advantages and disadvantages.Adult stem cells can be established from most tissues of the body, and the most frequently isolated ones come from bone marrow and adipose tissues of young and adult individuals and are referred to as “mesenchymal stem cells” (MSCs).Each of the cell types mentioned in Figure1 have different technical requirements for producing appropriate cells that could be used as therapeutic agents.Embryonic stem cells that are isolated from early-stage embryo present the particularity of being pluripotent and have an advantage over those cells from adult mesenchymal stem cells, which can differentiate only into a restricted number of cell lineages. However, cultures of these stem cell types are technically very demanding, because the amount of tissue to begin is very low for embryonic stem cells and the isolation of mesenchymal stem cells from the tissue mass is difficult (only 1 stem cell for every 104-5 cells in total adult tissue). Maintenance and expansion of stem cells in an undifferentiated state require the addition of many specific growth factors [28, 29], and so far, culture of embryonic stem cells and some mesenchymal stem cells are not possible without feeder layers which is in some part responsible for the inconsistent colony cell growth [29]. The necessity to use many exogenous growth factors as well as feeder layers to differentiate into specific cellular lineages are limiting factors for the scaleup of stem cell cultures for clinical applications. There are other major issues with these stem cell types for security as the cells can dedifferentiate once placed into an in vivo environment and even develop into tumors. Many techniques involving cell cloning or encapsulation would be necessary for assuring delivery of correct cell populations.Unlike stem cells, fetal cells are differentiated cells with high expansion, regeneration, and low immunogenic properties [10, 12]. As the fetal cells are already differentiated and do not need to be directed or altered, the vast number of additional growth factors normally necessary are not needed for cell culture and expansion, and these cells are not known to dedifferentiate once placed into the in vivoenvironment [7–9, 12].Establishment of cell banks is a crucial step in the process of many vaccines, medicinal products, or tissue-engineering products, and therefore, the choice of cell type is extremely important for technical and security reasons. A “cell bank” is the stocked product of consistent cell cultures that are frozen into small vials that withstand long-term freezing in liquid nitrogen (−165°C). The initial cell bank is frequently termed the master cell bank (MCB) from which each vial can derive a working cell bank (WCB). Whole cell bioprocessing and adaptable procedures to good manufacturing processes (GMPs) make it possible to develop extensive MCB and WCB to facilitate thorough testing of the cells. Once MCBs are accomplished, WCB can be produced to establish individual batches of treatments for high numbers of patients. Further, these cell banks can be tested completely for safety regarding sterility, pathogens and adventitious agents, and tumorigenicity.Historically, fetal cells have been used in cell banking procedures for medicinal products for many years already. Already in the 1930s, medical doctors and scientists have used tissue from voluntary pregnancy interruptions not only for understanding cell biology but also an important entity in the development of vaccines by using defined tissue-derived cell lines. The Nobel Prize for Medicine in 1954 was awarded to American immunologists who developed the polio vaccine based on cultures of human fetal cells. Since this time, many other necessary vaccines (rubella, chicken pox, hepatitis A, etc.) have been developed with the use of fetal cell lines including two primary human diploid cell lines which were originally prepared in the 1960s. The first cell line, WI-38 (Wistar Institute 38), was developed by Leonard Hayfleck in 1964 from fetal tissue from a voluntary pregnancy interruption and later given the ATCC (American Type Culture Collection) number of CCL-75. This cell line was used for the historical production of vaccine RA 27/3 against Rubella.


### 1.2. Fetal Wound Healing

Considerable interest and research have been dedicated to the understanding of wound healing and the associated process. Whereas adult cutaneous wounds heal more slowly and with scar formation to restore tissue integrity, fetal skin,in utero, is observed to have rapid and scarless tissue repair characterized by regeneration of an organized dermis with normal appendages and by a relative lack of inflammation [42–45]. Fundamental differences between fetal and adult skin and the fetal and adult skin wound environment may be important in inducing efficient tissue repair.Molecular analysis of wound healing has largely been devoted to cytokines and most particularly those of the transforming growth factor (TGF) family and their role in manipulating cutaneous wound healing and scar formation [46]. It has been suggested that scarless wound healing in fetal skin at early gestation is a result of the unique cytokine or growth factor profile [3, 45]. Of these, transforming growth factor-beta (TGF-β) has been most widely studied as it is implicated in the transition between scarless healing and repair with scar formation [47].Three highly homologous TGF-β isoforms are known in humans: β1, β2, and β3 [46]. Each form has been found by immunohistochemistry in unwounded fetal skin. However, low levels of TGF-β1 and high levels of TGF-β3 are expressed at gestational ages associated with scarless repair [3, 45].Exogenous application of TGF-β1 to normally scarless fetal wounds resulted in scar formation with an adult-like inflammatory response observed [47]. The profibrotic nature of TGF-β1, and possibly TGF-β2, was confirmed in wounds of adult rats, since neutralizing TGF-β1 and β2 with antibodies partially reduced the amount of scarring [48]. However, antifibrotic properties can be seen with the isoform TGF-β3 as injection, or application of this isoform showed reduced scarring and inflammation in adult wounds [48, 49].  When using a rabbit hypertrophic scar model, TGF-β3 was confirmed to show increased properties in wound healing but not scar reduction [50]. It has been suggested that the relative proportion of TGF-β isoforms, and not the absolute concentration of any one isoform determines the wound repair outcome [3, 45–47].TGF-β2 has been shown to be significantly higher in fetal skin dermal fibroblasts than in foreskin tissue fibroblasts but no major differences with TGF-β1 and TGF-β3 [11]. When looking at expression of the three isoforms in five different fetal skin cell lines, it was observed that TGF-β1 gene expression is much higher than that of TGF-β3. Importantly, variability between fetal donors for the TGF-β isoforms was very small which is generally the opposite for that of skin from young and old donors. Importance has been given to the TGF-β3 isoform by a company in England, Renovo Ltd., who have used human recombinant TGF-β3 (Juvista) in clinical trials showing 70% response rate for scar reduction to date.However, TGF-β1 and β2 neutralizing antibodies do not entirely prevent scarring in the adult, and other studies question the efficacy of TGF-β3 in wound healing [50].More recently, inhibition of TGFbRII-mediated signaling was demonstrated with a gene-therapy approach in a rabbit hypertrophic scarring model showing some reduction biologically. Lack of complete reduction could either be due to the technical difficulties suggested by the authors for low transduction efficiency, or it may suggest that factors other than TGF-β may also be important in scarless repair. As TGF-β’s particular importance in wound healing is also due to their ability to modulate ECM formation, two genes have previously been shown in wound healing in fetal skin (laminin b1, LAMb1) and in hypertrophic scar (dermatopontin, DPT) [51]. Dermatopontin and laminins have important roles in cell-matrix interactions and matrix assembly, and dermatopontin has been shown to be decreased in hypertrophic scar and systemic sclerosis skin fibroblasts [52].As wound healing is very complex, there are certainly many other molecules within the TGF-β superfamily which could have a role.  For instance, bone morphogenic protein (BMP) family of genes and their receptors are among those in the TGF-β superfamily genes and have also been strongly associated with cutaneous wound healing and scarless wound healing in the fetus. Overexpression of BMP-6 was shown to delay re-epitheliazation and promote scar formation in a transgenic mouse model [53]. BMP-6 is important for maintaining skin homeostasis and is 3.8 times higher expressed in fetal dermal fibroblasts than in newborn foreskin fibroblasts. More recent studies have shown evidence for the importance of angiogenesis and nerve involvement in wound repair [54, 55]. Pleiotrophin (PTN), a cytokine inducing heparin-binding/differentiation, is certain to have a major role in angiogenesis in wound healing. Midkine (MDK) and PTN, which have 50% amino acid sequence identity and striking domain homology, are the two members of the Ptn/Mdk developmental gene family [55].  Interestingly, PTN has been recently shown to induce functional neovasculature in vivo [55]. As fetal tissue heals with no inflammatory response, lower MDK and PTN expression is perhaps preferable, as they could have an important role in angiogenesis.In the past, gene profiling differences have been shown for fetal cells compared to adult cells [9], and therefore, it was also of interest to determine gene expression alterations in banked fetal dermal skin cells (used in tissue engineering for burns and wounds to date) and young dermal skin cells that have been banked in the same manner to have a listing of potential gene families from fetal banked cells to compare to young skin cells and other gene profiling studies. Young skin was, therefore, obtained from behind the ears of a 12 year old boy (12 yOS) following a surgical ear alignment and with the approval of the Hospital Ethics Committee to use this discarded material for research with parent and child oral and written approval. To identify differentially expressed genes in banked fetal dermal skin cells, we used cDNA microarray containing approximately 12,500 sequences (U95A human genome chip, Affymetrix UK, High Wycombe). Three arrays were hybridized for banked fetal skin cells (14 wFS) and banked young skin cells each (12 yOS). Briefly, RNA was isolated from cultured cells at passage one for each of the three cell banks. The statistical analyses were performed with a classical analysis of variance (ANOVA) followed by the global error assessment (GEA). Genes were selected by this method at α level 0.001. Significant genes, with at least a 1.5 fold change, were classified according to the gene ontology following the criteria of the DAVID (database for annotation, visualization, and integrated discovery) as described previously [9].When comparing banked fetal dermal skin cells to banked young fibroblasts with our conditions, 167 genes changed by 1.5 fold or more. Between those genes, 74 were upregulated in fetal cells and 93 were downregulated (Table1). Gene ontology of important differentially expressed genes was annotated following the criteria of the DAVID database (http://david.abcc.ncifcrf.gov/) for annotation, visualization, and integrated discovery) [56, 57]. As many of the genes analyzed could be involved in multiple biological processes, they have been placed in a category for their best representation (Figure 2).Table 1
Regulated genes in fetal banked cells compared to young banked cells.
Enter gene IDGene symbolGene nameFold increase (expressed in log2)3205HOXA9Homeobox A94.1032890GRIA1Glutamate receptor, ionotropic, AMPA 14.0503223HOXC6Homeobox C63.2553202HOXA5Homeobox A53.2432861GPR37G protein-coupled receptor 37 (Endothelin receptor type B-LIKE)3.2215789PTPRSProtein tyrosine phosphatase, receptor type, D3.1713206HOXA10Homeobox A103.0842321FLT1FMS-related tyrosine kinase 13.0609452ITM2AIntegral membrane protein 2A3.030862RUNX1T1RUNT-related transcription factor 1; translocated TO, 1 (cyclin D-related)2.9851047CLGNCalmegin2.916744MPPED2Metallophosphoesterase domain containing 22.8449056SLC7A7Solute carrier family 7, Member 72.8061948EFNB2Ephrin-B22.77023266LPHN2Latrophilin 22.7149865KIAA0644KIAA0644 gene product2.68923705CADM1Immunoglobulin superfamily, member 42.6071016CDH18Cadherin 18, type 22.5532294FOXF1Forkhead box F12.543140462ASB9DKFZP564L0862 protein2.4525947RBP1Retinol binding protein 1, cellular2.39483939EIF2AEukaryotic translation initiation factor 2A, 65 KDA2.2765918RARRES1Retinoic acid receptor responder (tazarotene induced) 12.2563306HSPA2Heat shock 70 KDA protein 22.2115880RAC2RAS-related C3 botulinum toxin substrate 2 (RHO family, small GTP binding protein RAC2)2.175151230KLHL23Kelch-like 232.171284ANGPT1Angiopoietin 12.1712201FBN2Fibrillin 2 (congenital contractural arachnodactyly)2.1643880KRT19Keratin 192.16357157PHTF2Putative homeodomain transcription factor 22.147154796AMOTAngiomotin2.1211012CDH13Cadherin 13, H-cadherin (heart)2.0865507PPP1R3CProtein phosphatase 1, regulatory (inhibitor) subunit 3C2.0421908EDN3Endothelin 32.0326319SCDStearoyl-coa desaturase (delta-9-desaturase)2.0225307PITX1Paired-like homeodomain transcription factor 12.0086899TBX1T-box 11.9575099PCDH7BH-protocadherin (brain-heart)1.9393215HOXB5Homeobox B51.8806664SOX11Sry (sex determining region Y)-box 111.8624004LMO1Lim domain only 1 (rhombotin 1)1.83010924SMPDL3ASphingomyelin phosphodiesterase, acid-like 3A1.8103233HOXD4Homeobox D41.8103232HOXD3Homeobox D31.8091305COL13A1Collagen, type XIII, alpha 11.772444ASPHAspartate beta-hydroxylase1.7675781PTPN11Protein tyrosine phosphatase, non-receptor type 11 (noonan syndrome 1)1.7494908NTF3Neurotrophin 31.7417046TGFBR1Transforming growth factor, beta receptor I (activin a receptor type II-like kinase, 53 KDA)1.7225743PTGS2Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)1.70823475QPRTQuinolinate phosphoribosyltransferase1.704934CD24CD24 antigen1.69810773ZBTB6Zinc finger protein 4821.6852048EPHB2EPH receptor B21.62423089PEG10Paternally expressed 101.61726013L3MBTLLethal (3) malignant brain tumor L(3)MBT protein (drosophila) homolog1.6143434IFIT1Interferon-induced protein with tetratricopeptide repeats 11.6135156PDGFRAPlatelet-derived growth factor receptor, alpha polypeptide1.610382ARF6ADP-ribosylation factor 61.6102028ENPEPGlutamyl aminopeptidase (aminopeptidase A)1.60710643IGF2BP3Insulin-like growth factor 2 MRNA binding protein 31.6048091HMGA2High mobility group at-hook 21.580900CCNG1Cyclin G11.5794192MDKMidkine (neurite growth-promoting factor 2)1.577552889LOC552889Hypothetical LOC5528891.576540ATP7BAtpase, CU++ transporting, beta polypeptide1.5652289FKBP5FK506 binding protein 51.5434194MDM4MDM4, transformed 3t3 cell double minute 41.5385764PTNPleiotrophin1.5338935SKAP2SRC family associated phosphoprotein 21.528273AMPHAmphiphysin1.5272799GNSGlucosamine (N-acetyl)-6-sulfatase (sanfilippo disease IIID)1.5195311PKD2Polycystic kidney disease 2 (autosomal dominant)1.51810402ST3GAL6ST3 beta-galactoside alpha-2,3-sialyltransferase 61.5013764KCNJ8Potassium inwardly-rectifying channel, subfamily J, member 8−1.5016347CCL2Chemokine (C-C motif) ligand 2−1.5092030SLC29A1Solute carrier family 29−1.5172919CXCL1Chemokine (C-X-C MOTIF) ligand 1−1.5191839HBEGFHeparin-binding EGF-like growth factor−1.5223488IGFBP5Insulin-like growth factor binding protein 5−1.5395678PSG9Pregnancy specific beta-1-glycoprotein 9−1.5573643INSRInsulin receptor−1.56910974C10ORF116Chromosome 10 open reading frame 116−1.5804071TM4SF1Transmembrane 4 L six family member 1−1.5801832DSPDesmoplakin−1.5904487MSX1MSH homeobox homolog 1−1.61327295PDLIM3PDZ and LIM domain 3−1.6151294COL7A1Collagen, type VII, alpha 1−1.617182JAG1Jagged 1 (alagille syndrome)−1.61722891ZNF365Zinc finger protein 365−1.6194223MEOX2Mesenchyme homeobox 2−1.6608076MFAP5Microfibrillar associated protein 5−1.66551363GALNAC4S-6STKIAA0598 gene product−1.6765354PLP1Proteolipid protein 1−1.6941410CRYABCrystallin, alpha B−1.6994854NOTCH3Notch homolog 3−1.743116039OSR2ODD-skipped related 2−1.75310873ME3Malic enzyme 3, NADP(+)-dependent, mitochondrial−1.7605865RAB3BRAB3B, member RAS oncogene family−1.76410391CORO2BCoronin, actin binding protein, 2B−1.77022809ATF5Activating transcription factor 5−1.7826356CCL11Chemokine (C-C motif) ligand 11−1.7837869SEMA3BSemaphorin 3B−1.8028404SPARCL1SPARC-like 1 (MAST9, HEVIN)−1.8131004CDH6Cadherin 6, type 2, K-cadherin (fetal kidney)−1.823131578LRRC15Leucine rich repeat containing 15−1.8363855KRT7Keratin 7−1.84611145HRASLS3HRAS-like suppressor 3−1.8535919RARRES2Retinoic acid receptor responder−1.8625493PPLPeriplakin−1.88227063ANKRD1Ankyrin repeat domain 1 (cardiac muscle)−1.891684BST2Bone marrow stromal cell antigen 2−1.9188190MIAMelanoma inhibitory activity−1.9327042TGFB2Transforming growth factor, beta 2−1.9506004RGS16Regulator of G-protein signalling 16−1.9697020TFAP2ATranscription factor AP-2 alpha−2.0083589IL11Interleukin 11−2.0228549LGR5Leucine-rich repeat-containing G protein-coupled receptor 5−2.0272277FIGFC-FOS induced growth factor−2.0294958OMDOsteomodulin−2.04710351ABCA8ATP-binding cassette, sub-family A (ABC1), member 8−2.0512065ERBB3V-ERB-B2 erythroblastic leukemia viral oncogene−2.0735649RELNReelin−2.1306663SOX10SRY (sex determining region Y)-BOX 10−2.1722878GPX3Glutathione peroxidase 3 (plasma)−2.1831620DBC1Deleted in bladder cancer 1−2.2084629MYH11Myosin, heavy polypeptide 11, smooth muscle−2.2214046LSP1Lymphocyte-specific protein 1−2.2245730PTGDSProstaglandin D2 synthase 21 KDA (BRAIN)−2.2526781STC1Stanniocalcin 1−2.2901675CFDComplement factor D (ADIPSIN)−2.2983490IGFBP7Insulin-like growth factor binding protein 7−2.31710439OLFM1Olfactomedin 1−2.34123090ZNF423Zinc finger protein 423−2.3721842ECM2Extracellular matrix protein 2−2.42611098PRSS23Protease, serine, 23−2.4317857SCG2SECRETOGRANIN II (CHROMOGRANIN C)−2.4482628GATMGlycine amidinotransferase−2.463358AQP1Aquaporin 1−2.5078418CMAHCytidine monophosphate-N-acetylneuraminic acid hydroxylase−2.5332006ELNElastin (supravalvular aortic stenosis, williams-beuren syndrome)−2.5893481IGF2Insulin-like growth factor 2 (somatomedin A)−2.5924804NGFRNerve growth factor receptor (tnfr superfamily, member 16)−2.6252675GFRA2GDNF family receptor alpha 2−2.646249ALPLAlkaline phosphatase, liver/bone/kidney−2.6712596GAP43Growth associated protein 43−2.67811075STMN2Stathmin-like 2−2.6955803PTPRZ1Protein tyrosine phosphatase, receptor-type, Z polypeptide 1−2.6958744TNFSF9Tumor necrosis factor superfamily, member 9−2.7238644AKR1C3Aldo-keto reductase family 1, member C3−2.8378839WISP2WNT1 inducible signaling pathway protein 2−2.910124ADH1CAlcohol dehydrogenase 1A (CLASS I)−2.9271734DIO2Deiodinase, iodothyronine, type II−2.9471116CHI3L1Chitinase 3-like 1 (cartilage glycoprotein-39)−3.3295320PLA2G2APhospholipase A2, group IIA (platelets, synovial fluid)−3.3553123HLA-DRB6Major histocompatibility complex, class II, DR beta 1−3.4153128HLA-DRB5Major histocompatibility complex, class II, DR beta 6−3.4154915NTRK2Neurotrophic tyrosine kinase, receptor, type 2−3.5523479IGF1Insulin-like growth factor 1 (somatomedin C)−3.6823897L1CAML1 cell adhesion molecule−3.706347APODApolipoprotein D−3.74950486G0S2G0/G1switch 2−3.9132824GPM6BGlycoprotein M6B−4.0291299COL9A3Collagen, type IX, alpha 3−4.0769244CRLF1Cytokine receptor-like factor 1−4.4632662GDF10Growth differentiation factor 10−4.8131311COMPCartilage oligomeric matrix protein−4.840Gene profiling and biological processes of fetal versus young skin cells. A cDNA microarray containing approximately 12,500 sequences (U95A human genome chip, Affymetrix UK, High Wycombe) was used to identify differentially expressed genes in banked fetal dermal skin cells compared to young skin cells. Three arrays were hybridised for each separate cell bank, and gene ontology of important differentially expressed genes was annotated following the criteria of the DAVID database (http://david.abcc.ncifcrf.gov/) for annotation, visualization, and integrated discovery. Biological processes of gene ontology is reported in the pie graphs for both up- and down-regulated genes (category for their best representation).Although individual growth factors (TGF-β2, TGF-β3, IL-10, and PDGF) have been shown in the clinic to help in different aspects of overall wound healing, it is indeed a very complex process [3, 45]. Most likely, many factors taken together are necessary for complete wound closure which could indeed be offered by a cell-based therapy for which many factors could be different in fetal cells such as those found in Table 1.Indeed, it has been shown that efficient repair could be obtained in 2nd and 3rd degree burns in children [7], acute wounds [8], and in chronic wounds [9] to date, and the whole-cell bioprocessing and age of the cells were very important aspects [10, 12].


## 1.1. Cellular Sources as Therapeutic Agents: Terminology Clarification, Technical Requirements, and Cell Banking

There is some confusion between the terminology and potential of embryonic, fetal, and adult stem cells. These cells are referred to in the literature as embryonic stem cells, fetal cells, and mesenchymal stem cells, respectively. However, more frequently, all of these cell types are referred to as simply stem cells, neglecting all of the legal and technical aspects associated with each specific cell type.  To illustrate these differences, Figure1 lists the major cell sources used in developing therapeutic applications showing that some cell choices are more adaptable to cellular therapy in patients. This adaptability is highly associated with technical facility of expanding and selecting cell populations needed. Tissue choices from animal and human at all ages of development can be evaluated with advantages and disadvantages for each final cell type (Figure 1).  In legal aspects, the term “embryo” denotes the earliest stages following fertilization of an ovum by a sperm. Zygote would include early stage cleavage embryos produced by cell division up to 50–60 cell stage (each cell which is a blastomere) and the blastocyte for the 60 cell stage to the point of implantation at about 2 weeks after-fertilization. “Embryonic stem cells” (ES) are developed from preimplantation embryos from the inner-cell mass before the first 2 weeks of development. These cells are frequently obtained from extra embryos developed by “in vitro” fertilization techniques to aid couples for fertility purposes. Because these particular cells have created quite an ethical debate, other researchers have begun using “fetal embryonic cells” derived from voluntary interruption of pregnancy between 5 and 8 weeks. Cell lines are normally developed from the genital ridge of the fetus. As this tissue is considered as an organ donation in most countries, it bypasses the major problems that have been raised by embryonic stem cells. Most fetal cell research is developed from specific tissues such as skin, muscle bone, or cartilage at the latter end of the first trimester (11–14 weeks) following voluntary interruption of pregnancies. Cell lines at this stage are tissue specific, and therefore, “fetal cells” are differentiated with specific functions. Fetal cells are derived from a legal organ donation when the mother donor ensures informed consent, provided that the tissue is a donation and not paid for and that there is no change in timing or method of pregnancy interruption for the sake of research. As fetal cells are from organ donations and under transplantation medicine, they are also routinely included in the terminology as adult stem cells.Figure 1
Cellular source and development stage with advantages and disadvantages.Adult stem cells can be established from most tissues of the body, and the most frequently isolated ones come from bone marrow and adipose tissues of young and adult individuals and are referred to as “mesenchymal stem cells” (MSCs).Each of the cell types mentioned in Figure1 have different technical requirements for producing appropriate cells that could be used as therapeutic agents.Embryonic stem cells that are isolated from early-stage embryo present the particularity of being pluripotent and have an advantage over those cells from adult mesenchymal stem cells, which can differentiate only into a restricted number of cell lineages. However, cultures of these stem cell types are technically very demanding, because the amount of tissue to begin is very low for embryonic stem cells and the isolation of mesenchymal stem cells from the tissue mass is difficult (only 1 stem cell for every 104-5 cells in total adult tissue). Maintenance and expansion of stem cells in an undifferentiated state require the addition of many specific growth factors [28, 29], and so far, culture of embryonic stem cells and some mesenchymal stem cells are not possible without feeder layers which is in some part responsible for the inconsistent colony cell growth [29]. The necessity to use many exogenous growth factors as well as feeder layers to differentiate into specific cellular lineages are limiting factors for the scaleup of stem cell cultures for clinical applications. There are other major issues with these stem cell types for security as the cells can dedifferentiate once placed into an in vivo environment and even develop into tumors. Many techniques involving cell cloning or encapsulation would be necessary for assuring delivery of correct cell populations.Unlike stem cells, fetal cells are differentiated cells with high expansion, regeneration, and low immunogenic properties [10, 12]. As the fetal cells are already differentiated and do not need to be directed or altered, the vast number of additional growth factors normally necessary are not needed for cell culture and expansion, and these cells are not known to dedifferentiate once placed into the in vivoenvironment [7–9, 12].Establishment of cell banks is a crucial step in the process of many vaccines, medicinal products, or tissue-engineering products, and therefore, the choice of cell type is extremely important for technical and security reasons. A “cell bank” is the stocked product of consistent cell cultures that are frozen into small vials that withstand long-term freezing in liquid nitrogen (−165°C). The initial cell bank is frequently termed the master cell bank (MCB) from which each vial can derive a working cell bank (WCB). Whole cell bioprocessing and adaptable procedures to good manufacturing processes (GMPs) make it possible to develop extensive MCB and WCB to facilitate thorough testing of the cells. Once MCBs are accomplished, WCB can be produced to establish individual batches of treatments for high numbers of patients. Further, these cell banks can be tested completely for safety regarding sterility, pathogens and adventitious agents, and tumorigenicity.Historically, fetal cells have been used in cell banking procedures for medicinal products for many years already. Already in the 1930s, medical doctors and scientists have used tissue from voluntary pregnancy interruptions not only for understanding cell biology but also an important entity in the development of vaccines by using defined tissue-derived cell lines. The Nobel Prize for Medicine in 1954 was awarded to American immunologists who developed the polio vaccine based on cultures of human fetal cells. Since this time, many other necessary vaccines (rubella, chicken pox, hepatitis A, etc.) have been developed with the use of fetal cell lines including two primary human diploid cell lines which were originally prepared in the 1960s. The first cell line, WI-38 (Wistar Institute 38), was developed by Leonard Hayfleck in 1964 from fetal tissue from a voluntary pregnancy interruption and later given the ATCC (American Type Culture Collection) number of CCL-75. This cell line was used for the historical production of vaccine RA 27/3 against Rubella.


## 1.2. Fetal Wound Healing

Considerable interest and research have been dedicated to the understanding of wound healing and the associated process. Whereas adult cutaneous wounds heal more slowly and with scar formation to restore tissue integrity, fetal skin,in utero, is observed to have rapid and scarless tissue repair characterized by regeneration of an organized dermis with normal appendages and by a relative lack of inflammation [42–45]. Fundamental differences between fetal and adult skin and the fetal and adult skin wound environment may be important in inducing efficient tissue repair.Molecular analysis of wound healing has largely been devoted to cytokines and most particularly those of the transforming growth factor (TGF) family and their role in manipulating cutaneous wound healing and scar formation [46]. It has been suggested that scarless wound healing in fetal skin at early gestation is a result of the unique cytokine or growth factor profile [3, 45]. Of these, transforming growth factor-beta (TGF-β) has been most widely studied as it is implicated in the transition between scarless healing and repair with scar formation [47].Three highly homologous TGF-β isoforms are known in humans: β1, β2, and β3 [46]. Each form has been found by immunohistochemistry in unwounded fetal skin. However, low levels of TGF-β1 and high levels of TGF-β3 are expressed at gestational ages associated with scarless repair [3, 45].Exogenous application of TGF-β1 to normally scarless fetal wounds resulted in scar formation with an adult-like inflammatory response observed [47]. The profibrotic nature of TGF-β1, and possibly TGF-β2, was confirmed in wounds of adult rats, since neutralizing TGF-β1 and β2 with antibodies partially reduced the amount of scarring [48]. However, antifibrotic properties can be seen with the isoform TGF-β3 as injection, or application of this isoform showed reduced scarring and inflammation in adult wounds [48, 49].  When using a rabbit hypertrophic scar model, TGF-β3 was confirmed to show increased properties in wound healing but not scar reduction [50]. It has been suggested that the relative proportion of TGF-β isoforms, and not the absolute concentration of any one isoform determines the wound repair outcome [3, 45–47].TGF-β2 has been shown to be significantly higher in fetal skin dermal fibroblasts than in foreskin tissue fibroblasts but no major differences with TGF-β1 and TGF-β3 [11]. When looking at expression of the three isoforms in five different fetal skin cell lines, it was observed that TGF-β1 gene expression is much higher than that of TGF-β3. Importantly, variability between fetal donors for the TGF-β isoforms was very small which is generally the opposite for that of skin from young and old donors. Importance has been given to the TGF-β3 isoform by a company in England, Renovo Ltd., who have used human recombinant TGF-β3 (Juvista) in clinical trials showing 70% response rate for scar reduction to date.However, TGF-β1 and β2 neutralizing antibodies do not entirely prevent scarring in the adult, and other studies question the efficacy of TGF-β3 in wound healing [50].More recently, inhibition of TGFbRII-mediated signaling was demonstrated with a gene-therapy approach in a rabbit hypertrophic scarring model showing some reduction biologically. Lack of complete reduction could either be due to the technical difficulties suggested by the authors for low transduction efficiency, or it may suggest that factors other than TGF-β may also be important in scarless repair. As TGF-β’s particular importance in wound healing is also due to their ability to modulate ECM formation, two genes have previously been shown in wound healing in fetal skin (laminin b1, LAMb1) and in hypertrophic scar (dermatopontin, DPT) [51]. Dermatopontin and laminins have important roles in cell-matrix interactions and matrix assembly, and dermatopontin has been shown to be decreased in hypertrophic scar and systemic sclerosis skin fibroblasts [52].As wound healing is very complex, there are certainly many other molecules within the TGF-β superfamily which could have a role.  For instance, bone morphogenic protein (BMP) family of genes and their receptors are among those in the TGF-β superfamily genes and have also been strongly associated with cutaneous wound healing and scarless wound healing in the fetus. Overexpression of BMP-6 was shown to delay re-epitheliazation and promote scar formation in a transgenic mouse model [53]. BMP-6 is important for maintaining skin homeostasis and is 3.8 times higher expressed in fetal dermal fibroblasts than in newborn foreskin fibroblasts. More recent studies have shown evidence for the importance of angiogenesis and nerve involvement in wound repair [54, 55]. Pleiotrophin (PTN), a cytokine inducing heparin-binding/differentiation, is certain to have a major role in angiogenesis in wound healing. Midkine (MDK) and PTN, which have 50% amino acid sequence identity and striking domain homology, are the two members of the Ptn/Mdk developmental gene family [55].  Interestingly, PTN has been recently shown to induce functional neovasculature in vivo [55]. As fetal tissue heals with no inflammatory response, lower MDK and PTN expression is perhaps preferable, as they could have an important role in angiogenesis.In the past, gene profiling differences have been shown for fetal cells compared to adult cells [9], and therefore, it was also of interest to determine gene expression alterations in banked fetal dermal skin cells (used in tissue engineering for burns and wounds to date) and young dermal skin cells that have been banked in the same manner to have a listing of potential gene families from fetal banked cells to compare to young skin cells and other gene profiling studies. Young skin was, therefore, obtained from behind the ears of a 12 year old boy (12 yOS) following a surgical ear alignment and with the approval of the Hospital Ethics Committee to use this discarded material for research with parent and child oral and written approval. To identify differentially expressed genes in banked fetal dermal skin cells, we used cDNA microarray containing approximately 12,500 sequences (U95A human genome chip, Affymetrix UK, High Wycombe). Three arrays were hybridized for banked fetal skin cells (14 wFS) and banked young skin cells each (12 yOS). Briefly, RNA was isolated from cultured cells at passage one for each of the three cell banks. The statistical analyses were performed with a classical analysis of variance (ANOVA) followed by the global error assessment (GEA). Genes were selected by this method at α level 0.001. Significant genes, with at least a 1.5 fold change, were classified according to the gene ontology following the criteria of the DAVID (database for annotation, visualization, and integrated discovery) as described previously [9].When comparing banked fetal dermal skin cells to banked young fibroblasts with our conditions, 167 genes changed by 1.5 fold or more. Between those genes, 74 were upregulated in fetal cells and 93 were downregulated (Table1). Gene ontology of important differentially expressed genes was annotated following the criteria of the DAVID database (http://david.abcc.ncifcrf.gov/) for annotation, visualization, and integrated discovery) [56, 57]. As many of the genes analyzed could be involved in multiple biological processes, they have been placed in a category for their best representation (Figure 2).Table 1
Regulated genes in fetal banked cells compared to young banked cells.
Enter gene IDGene symbolGene nameFold increase (expressed in log2)3205HOXA9Homeobox A94.1032890GRIA1Glutamate receptor, ionotropic, AMPA 14.0503223HOXC6Homeobox C63.2553202HOXA5Homeobox A53.2432861GPR37G protein-coupled receptor 37 (Endothelin receptor type B-LIKE)3.2215789PTPRSProtein tyrosine phosphatase, receptor type, D3.1713206HOXA10Homeobox A103.0842321FLT1FMS-related tyrosine kinase 13.0609452ITM2AIntegral membrane protein 2A3.030862RUNX1T1RUNT-related transcription factor 1; translocated TO, 1 (cyclin D-related)2.9851047CLGNCalmegin2.916744MPPED2Metallophosphoesterase domain containing 22.8449056SLC7A7Solute carrier family 7, Member 72.8061948EFNB2Ephrin-B22.77023266LPHN2Latrophilin 22.7149865KIAA0644KIAA0644 gene product2.68923705CADM1Immunoglobulin superfamily, member 42.6071016CDH18Cadherin 18, type 22.5532294FOXF1Forkhead box F12.543140462ASB9DKFZP564L0862 protein2.4525947RBP1Retinol binding protein 1, cellular2.39483939EIF2AEukaryotic translation initiation factor 2A, 65 KDA2.2765918RARRES1Retinoic acid receptor responder (tazarotene induced) 12.2563306HSPA2Heat shock 70 KDA protein 22.2115880RAC2RAS-related C3 botulinum toxin substrate 2 (RHO family, small GTP binding protein RAC2)2.175151230KLHL23Kelch-like 232.171284ANGPT1Angiopoietin 12.1712201FBN2Fibrillin 2 (congenital contractural arachnodactyly)2.1643880KRT19Keratin 192.16357157PHTF2Putative homeodomain transcription factor 22.147154796AMOTAngiomotin2.1211012CDH13Cadherin 13, H-cadherin (heart)2.0865507PPP1R3CProtein phosphatase 1, regulatory (inhibitor) subunit 3C2.0421908EDN3Endothelin 32.0326319SCDStearoyl-coa desaturase (delta-9-desaturase)2.0225307PITX1Paired-like homeodomain transcription factor 12.0086899TBX1T-box 11.9575099PCDH7BH-protocadherin (brain-heart)1.9393215HOXB5Homeobox B51.8806664SOX11Sry (sex determining region Y)-box 111.8624004LMO1Lim domain only 1 (rhombotin 1)1.83010924SMPDL3ASphingomyelin phosphodiesterase, acid-like 3A1.8103233HOXD4Homeobox D41.8103232HOXD3Homeobox D31.8091305COL13A1Collagen, type XIII, alpha 11.772444ASPHAspartate beta-hydroxylase1.7675781PTPN11Protein tyrosine phosphatase, non-receptor type 11 (noonan syndrome 1)1.7494908NTF3Neurotrophin 31.7417046TGFBR1Transforming growth factor, beta receptor I (activin a receptor type II-like kinase, 53 KDA)1.7225743PTGS2Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)1.70823475QPRTQuinolinate phosphoribosyltransferase1.704934CD24CD24 antigen1.69810773ZBTB6Zinc finger protein 4821.6852048EPHB2EPH receptor B21.62423089PEG10Paternally expressed 101.61726013L3MBTLLethal (3) malignant brain tumor L(3)MBT protein (drosophila) homolog1.6143434IFIT1Interferon-induced protein with tetratricopeptide repeats 11.6135156PDGFRAPlatelet-derived growth factor receptor, alpha polypeptide1.610382ARF6ADP-ribosylation factor 61.6102028ENPEPGlutamyl aminopeptidase (aminopeptidase A)1.60710643IGF2BP3Insulin-like growth factor 2 MRNA binding protein 31.6048091HMGA2High mobility group at-hook 21.580900CCNG1Cyclin G11.5794192MDKMidkine (neurite growth-promoting factor 2)1.577552889LOC552889Hypothetical LOC5528891.576540ATP7BAtpase, CU++ transporting, beta polypeptide1.5652289FKBP5FK506 binding protein 51.5434194MDM4MDM4, transformed 3t3 cell double minute 41.5385764PTNPleiotrophin1.5338935SKAP2SRC family associated phosphoprotein 21.528273AMPHAmphiphysin1.5272799GNSGlucosamine (N-acetyl)-6-sulfatase (sanfilippo disease IIID)1.5195311PKD2Polycystic kidney disease 2 (autosomal dominant)1.51810402ST3GAL6ST3 beta-galactoside alpha-2,3-sialyltransferase 61.5013764KCNJ8Potassium inwardly-rectifying channel, subfamily J, member 8−1.5016347CCL2Chemokine (C-C motif) ligand 2−1.5092030SLC29A1Solute carrier family 29−1.5172919CXCL1Chemokine (C-X-C MOTIF) ligand 1−1.5191839HBEGFHeparin-binding EGF-like growth factor−1.5223488IGFBP5Insulin-like growth factor binding protein 5−1.5395678PSG9Pregnancy specific beta-1-glycoprotein 9−1.5573643INSRInsulin receptor−1.56910974C10ORF116Chromosome 10 open reading frame 116−1.5804071TM4SF1Transmembrane 4 L six family member 1−1.5801832DSPDesmoplakin−1.5904487MSX1MSH homeobox homolog 1−1.61327295PDLIM3PDZ and LIM domain 3−1.6151294COL7A1Collagen, type VII, alpha 1−1.617182JAG1Jagged 1 (alagille syndrome)−1.61722891ZNF365Zinc finger protein 365−1.6194223MEOX2Mesenchyme homeobox 2−1.6608076MFAP5Microfibrillar associated protein 5−1.66551363GALNAC4S-6STKIAA0598 gene product−1.6765354PLP1Proteolipid protein 1−1.6941410CRYABCrystallin, alpha B−1.6994854NOTCH3Notch homolog 3−1.743116039OSR2ODD-skipped related 2−1.75310873ME3Malic enzyme 3, NADP(+)-dependent, mitochondrial−1.7605865RAB3BRAB3B, member RAS oncogene family−1.76410391CORO2BCoronin, actin binding protein, 2B−1.77022809ATF5Activating transcription factor 5−1.7826356CCL11Chemokine (C-C motif) ligand 11−1.7837869SEMA3BSemaphorin 3B−1.8028404SPARCL1SPARC-like 1 (MAST9, HEVIN)−1.8131004CDH6Cadherin 6, type 2, K-cadherin (fetal kidney)−1.823131578LRRC15Leucine rich repeat containing 15−1.8363855KRT7Keratin 7−1.84611145HRASLS3HRAS-like suppressor 3−1.8535919RARRES2Retinoic acid receptor responder−1.8625493PPLPeriplakin−1.88227063ANKRD1Ankyrin repeat domain 1 (cardiac muscle)−1.891684BST2Bone marrow stromal cell antigen 2−1.9188190MIAMelanoma inhibitory activity−1.9327042TGFB2Transforming growth factor, beta 2−1.9506004RGS16Regulator of G-protein signalling 16−1.9697020TFAP2ATranscription factor AP-2 alpha−2.0083589IL11Interleukin 11−2.0228549LGR5Leucine-rich repeat-containing G protein-coupled receptor 5−2.0272277FIGFC-FOS induced growth factor−2.0294958OMDOsteomodulin−2.04710351ABCA8ATP-binding cassette, sub-family A (ABC1), member 8−2.0512065ERBB3V-ERB-B2 erythroblastic leukemia viral oncogene−2.0735649RELNReelin−2.1306663SOX10SRY (sex determining region Y)-BOX 10−2.1722878GPX3Glutathione peroxidase 3 (plasma)−2.1831620DBC1Deleted in bladder cancer 1−2.2084629MYH11Myosin, heavy polypeptide 11, smooth muscle−2.2214046LSP1Lymphocyte-specific protein 1−2.2245730PTGDSProstaglandin D2 synthase 21 KDA (BRAIN)−2.2526781STC1Stanniocalcin 1−2.2901675CFDComplement factor D (ADIPSIN)−2.2983490IGFBP7Insulin-like growth factor binding protein 7−2.31710439OLFM1Olfactomedin 1−2.34123090ZNF423Zinc finger protein 423−2.3721842ECM2Extracellular matrix protein 2−2.42611098PRSS23Protease, serine, 23−2.4317857SCG2SECRETOGRANIN II (CHROMOGRANIN C)−2.4482628GATMGlycine amidinotransferase−2.463358AQP1Aquaporin 1−2.5078418CMAHCytidine monophosphate-N-acetylneuraminic acid hydroxylase−2.5332006ELNElastin (supravalvular aortic stenosis, williams-beuren syndrome)−2.5893481IGF2Insulin-like growth factor 2 (somatomedin A)−2.5924804NGFRNerve growth factor receptor (tnfr superfamily, member 16)−2.6252675GFRA2GDNF family receptor alpha 2−2.646249ALPLAlkaline phosphatase, liver/bone/kidney−2.6712596GAP43Growth associated protein 43−2.67811075STMN2Stathmin-like 2−2.6955803PTPRZ1Protein tyrosine phosphatase, receptor-type, Z polypeptide 1−2.6958744TNFSF9Tumor necrosis factor superfamily, member 9−2.7238644AKR1C3Aldo-keto reductase family 1, member C3−2.8378839WISP2WNT1 inducible signaling pathway protein 2−2.910124ADH1CAlcohol dehydrogenase 1A (CLASS I)−2.9271734DIO2Deiodinase, iodothyronine, type II−2.9471116CHI3L1Chitinase 3-like 1 (cartilage glycoprotein-39)−3.3295320PLA2G2APhospholipase A2, group IIA (platelets, synovial fluid)−3.3553123HLA-DRB6Major histocompatibility complex, class II, DR beta 1−3.4153128HLA-DRB5Major histocompatibility complex, class II, DR beta 6−3.4154915NTRK2Neurotrophic tyrosine kinase, receptor, type 2−3.5523479IGF1Insulin-like growth factor 1 (somatomedin C)−3.6823897L1CAML1 cell adhesion molecule−3.706347APODApolipoprotein D−3.74950486G0S2G0/G1switch 2−3.9132824GPM6BGlycoprotein M6B−4.0291299COL9A3Collagen, type IX, alpha 3−4.0769244CRLF1Cytokine receptor-like factor 1−4.4632662GDF10Growth differentiation factor 10−4.8131311COMPCartilage oligomeric matrix protein−4.840Gene profiling and biological processes of fetal versus young skin cells. A cDNA microarray containing approximately 12,500 sequences (U95A human genome chip, Affymetrix UK, High Wycombe) was used to identify differentially expressed genes in banked fetal dermal skin cells compared to young skin cells. Three arrays were hybridised for each separate cell bank, and gene ontology of important differentially expressed genes was annotated following the criteria of the DAVID database (http://david.abcc.ncifcrf.gov/) for annotation, visualization, and integrated discovery. Biological processes of gene ontology is reported in the pie graphs for both up- and down-regulated genes (category for their best representation).Although individual growth factors (TGF-β2, TGF-β3, IL-10, and PDGF) have been shown in the clinic to help in different aspects of overall wound healing, it is indeed a very complex process [3, 45]. Most likely, many factors taken together are necessary for complete wound closure which could indeed be offered by a cell-based therapy for which many factors could be different in fetal cells such as those found in Table 1.Indeed, it has been shown that efficient repair could be obtained in 2nd and 3rd degree burns in children [7], acute wounds [8], and in chronic wounds [9] to date, and the whole-cell bioprocessing and age of the cells were very important aspects [10, 12].


## 2. Fetal Tissue Collection, Culture, and Cell Bank Requirements

One of the major challenges for assuring that more patients will benefit from cell-based therapies in the future will be the optimisation of the choice of cell type as well as their isolation and proliferation. The development of master cell banks from the cell choice provides a major advantage for the creation of a therapeutic biological agent. Careful selection of donors and extensive screening of both the donor and cultured cells avoids transmissible viral, fungal, or bacterial disease and, therefore, can provide a safe and secure utilization of cells for therapeutic purposes.Fetal cells have the advantage of being an organ donation, and only very small biopsies are necessary for developing extensive, consistent master cell banks for many tissues. Thorough safety testing of mother donor for infectious diseases (status of the donor for HIV, HBV, and HCV) at the time of tissue donation and again 3 months after to ensure negative sero-conversion can be ensured. During the informed consent of the patient, an interview for overall health is recorded to evaluate eligibility into an organ donation program.  For the tissue, information concerning the age of the fetus, date, time, and place of acquirement need to be obtained along with types and amounts of tissue received. The transfer containers can be labelled with the identification code to assure an anonymous organ donation. All culture products used for the transfer of tissue and cell culture need to be of clinical grade quality. Cell banking and testing criteria need to be accomplished under cGMP conditions such as required for human diploid cells used for vaccine production and for cell substrates used for biotechnological products. Testing routinely accomplished on cell banks includes tests for viruses, virus-like particles, mycoplasmas, fungi, yeasts, and bacteria with bothin vitro and in vivo testing. Donor source is identified by isoenzyme testing, and in vitro testing of picornavirus, orthomyxovirus, pramyxovirus, herpesvirus, adenovirus, and reovirus is routinely accomplished with several control cell lines when the tissue is human source (Q-PCR for HepB, HepC, HIV-1, HIV-2, HTLV-1, HTLV-2, HHV-6, HHV-7, HHV-8, EBV, hCMV, SV40, and B19 parvovirus). In vivovirus testing using suckling mice, adult mice, guinea pigs and embryonated eggs is routinely employed.It is important to note that until now, there have been no reported biopharmaceuticals derived from continuous cell cultures that have been implicated in the transmission of infectious agents to humans. Most sources of contamination are adventitious, which means that the contamination is introduced from an external source such as the culture products.


## 3. Cellular Delivery Systems: Collagen Matrix, Cream, and Hydrogel

Once safety can be assured, efficient cell presentation with biocompatible delivery systems can be assessed for specific tissues. For delivery systems, biocompatible biomaterials need to be available in order to provide an extracellular matrix environment for cell differentiation, delivery, and release. To be admitted for clinical use, it is more rapid to use approved medical device quality material. Cell and materials need to be tested together to assure not only biocompatibility but also their interactions, cellular stability, possible degraded by-products of combination, and degradation or absorption. Ease of applicability of the final product will be of importance for clinical use.Preparations from biologicals, and particularly from live cells and their delivery to the patient are a major complication for treatment, and the shelf life is very limited with such preparations. Many products for skin repair have applied alternatives such as freeze-dried, frozen, and refrigerator stocking [18–23]. Stability and bioequivalence will have to meet the exigent and stringent technical aspects for development of therapeutic products.Cellular therapies can be delivered as three-dimensional constructs (tissue-engineered live cell constructs) that can be placed topically over wound and scar surfaces or in solutions composed of creams or biogels. For large-surface wounds and areas that are difficult to apply overlying bandages, cream and biogel preparations permit an advantage for multiple daily applications with either live (hydrogel) or inactivated (cream) cells (Figure3). Final preparations need to be consistent with equivalent “dosage” of cellular products delivered such as a cell density or by protein content.Figure 3
Cell delivery by “biological bandage”, hydrogel, and pharmaceutical cream formulation.  Each identical ampoule of fetal skin cells (working cell bank) can be seeded directly into biocompatible collagen (Tissue Fleece, Baxter, Switzerland), hydrogels (Mesolis, Anties, Plan-les-Ouates, Switzerland), or into pharmaceutical cream formulations (oil-in-water emulsion base was prepared under GLP, Good Laboratory Practices) that assure biological stability.


## 4. Assessment of Fetal Cell Therapy in Wound and Scar Management

Management of wounds using fetal cell therapy have been intended for both acute and chronic wounds and for burns. In the first clinical studies reported using continuous fetal skin cell lines [7, 8], they were designed to prepare wound beds with fetal cells before autografting of patients who were programmed for secondary surgery for wound closure. This fetal cell preparative step was accomplished with the intention of having both a better “take” for the autograft and enhanced quality of treated skin. For pediatric burn patients, the pretreatment with fetal cells showed to be very efficient in wound closure to the point, where all 8 patients included in the study did not have to have the planned autografts for continued treatment [7, 8]. Treated skin showed complete closure rapidly with little hypertrophy of new skin and no retraction seen.In studies reported using fetal cells for chronic leg ulcers, patients were selected on the basis of a history of having refractory chronic leg ulcers, which did not heal using traditional therapies, such as compression (active and passive), hydrocolloids, and autografts [9]. Ulcers such as illustrated in Figure 4 (female patient 1, with a history of painful postthrombotic ulcer for 14 years, consecutive to deep venous system and short saphenous vein insufficiency) portray the use of several delivery systems of live and inactivated fetal skin cells. For this particular patient, previous autografts and different compression therapies including 4 layers bandages were not successful. Immediately following the first fetal skin construct, edema diminished, pain was relieved, and fibrin production elimination was evident. Applications of fetal skin constructs one time per week were capable of gradual but rapid closure of this large, deep, and painful ulcer. At 11 weeks, the larger portion of the ulcer was healed. Healed ulcer and surrounding skin was treated with pharmaceutical cream preparation with inactivated fetal skin cells until full closure. At one year followup, the patient shows skin that is still atrophic but with no presence of scar tissue (Figure 4). Although etiologies of ulcer patients are varied, similar observations were reported using fetal skin cells for treatment in 8 patients representing 13 ulcers included in the clinical safety studies [9]. Examples of rapid evolution of acute wounds and burns treated in the same manner with fetal cell preparations are portrayed in Figure 5. These patients were selected to represent the early repair processes noted and associated anti-inflammatory effects. Regardless of wound etiology (Patient 2 presented with an incision from a glass wound, Patient 3 with an intense soder burn, and Patient 4 with an acid burn), it can be generally observed that rapid epitheliazation of skin was initiated.Fetal cell therapy of chronic wounds. Patient 1 (female 64 years old) with a history of painful postthrombotic ulcer for 14 years, consecutive to deep venous system, and short saphenous vein insufficiency (a). Evolution of the large, deep, and painful ulcer with applications of fetal skin constructs one time per week at 1 month (b) and two months (c).  Following the majority of closure at 2 months, surrounding skin was treated with pharmaceutical cream preparation with inactivated fetal skin cells until full closure at 11 weeks (d). At one year followup, the patient shows skin that is still atrophic but no presence of scar tissue. “Biological bandage” preparations: fetal cells (seeding density of 5×103 cells/cm2 with cells from the WCB at passages 3-4) were placed in culture media and seeded on the collagen sheet and placed into a 37°C incubator at 95% relative humidity and 10% CO2. Final products were employed for the patients in clinical Phase I and II studies for burns and wounds.Cream preparation: the cream was prepared under controlled, clean-room conditions in an automated pharmaceutical machine (Moltomat, Krieger AG, Basel, Switzerland). Its composition contained hydrogenated vegetable oil, glycerine, propylene glycol, cetearyl, ethyhexanoate, decyl oleate, ceteanyl alcohol, cetyl palmitate, glucose, ascorbyl palmitate, tocopheryl acetate, propylparaben, methylparaben, potassium chloride, magnesium chloride, sodium cetearyl sulphate, and simethicone. Samples were tested for quality control with respect to microbiological and physicochemical (viscosity, pH, conductivity, mass volume, colorimetry, and microscopy). Cell concentrations of 5×103/mL were used on deteriorated skin of patients surrounding chronic ulcers and following wound closure of wound burn patients (Figure 5).
(a)(b)(c)(d)Figure 5
Patients with various forms of acute wounds (Patient 2) glass incision (Patient 3) soder burn, and (Patient 4) acid burn treated with fetal cell preparations as described for Figure4. Evolution of wounds early in treatment with bandage changes every 2 days illustrates rapid wound repair with low associated inflammation.


## 5. Discussion

Biologicals for wound and scar management have predominantly been developed with neonatal or young foreskin tissue cell culture to date (companies including, Smith and Nephew, XCELLentis, Organogenesis, Ortec, and DFB Pharmaceuticals) [13–15, 17, 18]. Differences of expression in wound healing gene families between fetal cells and foreskin cells used in biological preparations could be responsible for more efficient repair processes seen in the clinic and particularly for the rapidity in the treatment of acute wounds and burns [11]. Fetal skin represents the ideal paradigm of all tissue repair due to its inherent ability to repair through regeneration rather than scar. Even though fetal wound repair is a tightly regulated process involving many cellular mediators, the precise mechanisms of efficient wound healing without scar formation remain unknown despite the great increase in knowledge gained over the past decades. Chen et al. [58] have proposed that understanding the “blueprint of fetal skin repair” might allow the manipulation of adult wound healing in order to decrease scarring and fibrosis. There are indeed many genes that are significantly different in the “fetal skin blueprint” when compared to both younger and older skin that have been elucidated in previous work [9] and in this paper with banked fetal and young skin cells accomplished with the same cell banking techniques. Herein, we show that expression profiling of banked fetal cells compared to young skin has provided biological grouping of important gene families implicated in the mechanism of wound repair (such as cell adhesion, angiogenesis, and development; see pie graphs Figure 2, Table 1).Indeed, individual growth factors (TGF-β2, TGF-β3, IL-10, and PDGF) have been shown in the clinic to help in different aspects of overall wound healing, but it is a very complex process. Most likely, many factors taken together are necessary for complete wound closure which could indeed be offered by a cell-based therapy for wound management. In making parallels with currently used medicines, it is now becoming more and more of a problem for single growth factors in wound and tissue repair. As milligram quantities are necessary for treatment of wounds such as that with platelet-derived growth factor (PDGF, Regranex), the long-term use is showing some safety concern. The use of this growth factor for the treatment of chronic leg ulcers has now been limited to short treatment regimens due to increased cancer in the patients (Ortho-McNeil-Janssen Pharmaceuticals; Important Drug Warning insert of medication). When only one growth factor or cytokine is used, the dosage accumulations are very high.  Cellular therapies (or total cellular products) could provide the more correct balance for dosages of individual proteins or factors. The pmol/nmol quantities of individual proteins (when delivered all together are ~2-3 μg) have been shown to be effective without the secondary effects on long term [7–9].New biologicals may be of high interest if the safety and simplicity can be assured and if the overall cost can be limited.Regarding the use of the cells for preclinical trials, it will be particularly important to ensure consistency of growth of the cells and consistency of the harvest obtained. High consistency in fetal cell banking can be achieved due to the minimum requirements of fetal cell cultures. In contrast with MSC, fetal cells do not require feeder layers for growth nor growth factors for differentiation. Fetal cells show qualities required for the establishment of GMP cell banks to be used for medicinal and tissue-engineering products. The lifespan and the proliferation rate of the fetal cells can allow master cell banks of 100–300 ampoules containing 5–10 millions cells each from 2 cm2 of tissue at very early passaging (Passage 1 and 2). MCB and WCB have been prepared from fetal skin tissue in short periods of time compared to other primary cells [8, 10].Regulation of final products and the simplicity will be mandatory milestones for biological development. Until 2007, Phase I and II safety studies with fetal cells were regulated in Switzerland (where the clinical trials were held) by the Department of Public Health (OFSP) under Transplantation Law (for living cell transplants) and by “hospital preparations in small quantities” by the state chemist (for inactivated cells). For comparison in the United States by the FDA, other cell types (i.e., foreskin) were regulated, in the majority, as medical devices or cosmetic products.  New regulatory requirements issued in recent years (2007) will assure better safety, but cost will be largely affected. All cellular products must be in compliance with guidelines of good manufacturing practice (GMP) with respect to medicinal products and investigational medicinal products for human use. The European Union (EU) regulation on advanced therapy medicinal products (ATMPs) was adopted in all European Member States on Dec. 30, 2008, and the FDA recently also proposed regulations on human cells, tissues, as well as for cellular and tissue-based products. The main scope of the regulations is to establish clear classification criteria for many new cell-based medicinal products. For the EU, it makes reference to the 2004/23/EC directive on donation, procurement, and testing of human cells and tissues and also with directive 2002/98/EC on human blood and blood components. All together, these directives dictate that human cells have to be in compliance with the quality requirements therein described and that all ATMP have to be prepared under GMP conditions. Key elements including identity, purity, sterility, stability, safety, and efficacy are recommended for cellular-based products. In all, these new regulations impose strict criteria for the production and the environment used for the production of cell-based products to be used in clinical trials and treatments [59–69]. Evolution of advanced cellular therapeutics worldwide and how they are regulated will have a major impact on availability to patients. Clear regulatory affairs of cellular use, whether they are delivered living or inactivated, will be necessary to help researchers and clinicians in future therapies.Cell therapies and tissue engineering are beginning to show great promise in wound and scar management. The cell choice is, therefore, an important factor for simplifying the overall technique and bringing therapy rapidly to the patient.Thus, fetal cells with their high expansion, simple culture conditions (do not require feeder layers or extensive growth factors for expansion which is a major reason for their consistency in scaling out), and low immunogenicity properties [10, 12] are ideal conditions for whole-cell bioprocessing destined for cell therapy, tissue-engineering, and medicinal products. Additionally, they have already been used in safety clinical Phase I and II studies showing rapid and efficient tissue repair with minimal scarring [7–9, 12].Delivery systems to afford better stocking and stability will be important milestones for biological products, and topical preparations that show biological activity would be a great benefit. Overall, the advantage of cellular preparations is that there is no need for a chemical “active ingredient” for wound healing and scar management.



---

*Source: 549870-2011-05-18.xml*