(JDD Jan 2023) Improvement in Skin Elasticity Using Red Deer Umbilical Cord Lining Mesenchymal Stem Cell Conditioned Media

Abstract

Background: Significant improvement in skin tone was reported after topical application of a facial cream (CALECIM®Professional Multi-Action Cream, CALECIM Cosmeceuticals, Singapore) containing conditioned media (CM) derived from Red Deer Umbilical Cord Lining Mesenchymal Stem Cell (RD-CLMSC) culture. This study investigates the paracrine effects of RD-CLMSC-CM on human dermal fibroblasts (HDF) to understand how it may increase skin turgor and elasticity.

Skin aging is associated with lower levels of extracellular matrix components such as hyaluronic acid (HA) and elastin, resulting in poor skin turgor and elasticity. Histochemical staining followed by photocolorimetry demonstrated that RD-CLMSC-CM upregulated HDF expression of elastin by 56% and HA by 83% compared with DMEM/10% Fetal Calf Serum (FCS).

To further quantify the effects of CM, aproliferation assay was used to assess HDF response to RD-CLMSC-CM exposure. Exposure to RD-CLMSC-CM resulted in the highest increase in HDF proliferation over DMEM/10% FCS (113%) followed by Human (H)-CLMSC-CM (112%), then Human Foreskin Fibroblast (FSF)-CM (16%).

These experimental results demonstrate both the cross-species efficacy and lack of toxicity of RD-CLMSC-CM on HDF.These pre­ clinical studies also suggest the clinical effects of RD-CLMSC-CM on skin turgor may be related to increased HA and elastin production by HDF. as well as enhanced proliferation.

Introduction

Stem cells were first isolated from the bone marrow.1 Since that time, they have also been found in adipose tissue, dental pulp, various parts of the placenta and umbilical cord, and embryos.2 The placenta and umbilical cord, which are often discarded after delivery, represent attractive stem cell sources given their ethical acceptability.

In 2004,Cord Lining Mesenchymal Stem Cells(CLMSC) andCord Lining Epithelial Stem Cells (CLEpSC) were isolated from the umbilical cord and found to be present in enormous quantities with extremely high purity.3 The cells were additionally found to express non-classical HLA subtypes, HLA-E and HLA-G, which conferred immune protection to these cells by suppressing rejection. Human Cord Lining Mesenchymal Stem Cells (H-CLMSC) seeded on a Biobrane (Smith and Nephew, Hull, UK) scaffold were used initially as a biological dressing to improve granulation tissue in burn patients prior to skin grafting. Accelerated wound re-epithelialisation and healing was observed. This led to the application of H-CLMSC to other chronic wounds which demonstrated accelerated wound healing (unpublished observations). Current Good Manufacturing Practice (cGMP) grade H-CLMSC cultured and expanded in a Biologics Licensing Application (BLA) facility at the University of Colorado Anschutz Medical Campus are currently being used in a United States Food and Drug Administration (USFDA) trial to accelerate wound healing in chronic diabetic foot ulcers.7 Given the positive effects of H-CLMSC on wound healing, it was postulated that they may also be beneficial in the rejuvenation of aged skin. Guided by the Association of South East Asian Nations (ASEAN) Cosmetic Directive, which prohibits cosmetic products containing derivatives of human origin, umbilical cords from Red Deer (RD) raised for horn velvet in New Zealand were harvested and CLMSC cultured for the collection of conditioned media (CM), which contains metabolites, growth factors and extracellular matrix proteins that have been shown to promote tissue repair,8•9 regenerate hair follicles,10•11 and accelerate wound healing through optimization of fibroblasts.12 Extracellular vesicles (EVs) are a heterogeneous collection of membrane-bound carriers including exosomes and microvesicles with complex cargos, including lipids, proteins, and nucleic acid, the contents of which contribute to the varied components of conditioned media. MSCs, secrete EVs to exert paracrine effects on cells that internalize the secreted EVs.13-17 A list of secretory cytokines and growth factors expressed by H-CLMSC-CM is found in Table 1.

The primary objective of this study is to investigate the expression of HA and elastin in HOF exposed to RD-CLMSC­ CM, as well as to assess HOF proliferation post exposure to RD-CLMSC-CM vs Human (H)-CLMSC-CM vs Human Foreskin Fibroblast conditioned medium (FSF-CM).

Materials and Methods

RD Umbilical Cords from New Zealand
RD umbilical cords were collected under specific handling instructions from a farm in New Zealand that rears the animal for collection of horn velvet.The cords were transported in DMEM/ Penicillin/Streptomycin/Amphotericin B between 2° and 8° C for the entire transfer duration from the farm to the laboratory in Singapore.

Isolation of RD-CLMSC
RD-CLMSC were isolated from umbilical cords according to the protocol described in international patent publication number WO 2006/019357 Al Briefly, the umbilical cords were washed in PBS supplemented with Penicillin/Streptomycin/Amphotericin B until the solution turned clear. The amniotic membrane was divided into small pieces, placed on the plastic surface of the culture flask, submerged in PTT-6 medium (CellResearch Corp, Singapore), and incubated at 37° C with 5% CO . The culture medium was changed every 2 to 3 days. At a confluence of about 70%, cells were trypsinized (0.05% trypsin/0.02% EDTA) for further expansion or for cryo-preservation.

HOF,Aged HOF,FSF,and H-CLMSC
HDFs were selected from the tissue bank of Cell Research Corporation (CRC) from sources listed in Table 2, and were divided into Normal Fibroblasts (NF) for individuals below 60 years of age and Aged Skin Fibroblasts (asF) for individuals 60 years and older.

H-CLMSC (#CLMC43) was also obtained from the CRC tissue bank. FSF was given to CRC as a gift from the Stem Cell and Wound Healing Research Group, Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore.

Conditioned Media Was Derived From RD-CLMSC, H-CLMSC, FSF Culture
Cryovials containing RD-CLMSC, H-CLMSC, and FSF were retrieved from storage and quick thawed in a 37° C water bath. PTT-6 medium (CellResearch Corporation, Singapore) was used for culturing CLMSC (RD & H), and DMEM with 10% FCS was used for culturing FSF, all at 37° C with 5% CO2. PTT-6 and DMEM/10% FCS were changed every 2 to 3 days. At 100% confluency, PTT-6 and DMEM/10% FCS were replaced with DMEM basal medium. Culture dishes were incubated for 48 hours and spent medium was collected into centrifuge tubes for centrifugation at 1800 rpm for 10 minutes. The supernatant was collected as conditioned medium (CM) into labeled tubes: Red Deer Cord Lining Mesenchymal Stem Cell Conditioned Medium (RD-CLMSC-CM), Human Cord Lining Mesenchymal Stem Cell Conditioned Medium (H-CLMSC-CM), and Human Foreskin

Fibroblast Conditioned Medium (FSF-CM). Conditioned media were kept at -80° C until use.

Total Cell Counts to Measure Proliferative Effects of H-CLMSC­ CM, RD-CLMSC-CM, FSF-CM, and DMEM/10% FCS on Aged

Skin HOF
Aged skin dermal fibroblasts (asF) were isolated from the skin of donors aged 60 years and above (asF74/ asF75/ asF76;Table 2). asF were seeded equally (in triplicate) into 24 well plates containing DMEM/10% FCS, and incubated at 37° C and 5% CO2 for 24 hours to allow cellular adherence. After 24 hours, medium was removed from the plates and the wells rinsed once with PBS. At day 0, HOF were cultured in DMEM/10% FCS (control), FSF-CM, H-CLMSC-CM, or RD-CLMSC-CM. Media was changed on day 2. On day 5, media was removed and the cells were rinsed once with PBS. Cells were trypsinized and observed under the microscope to ensure that all the cells were dislodged before neutralization by the addition of 1 ml DMEM/10% FCS. Cell suspensions were centrifuged at 1800rpm for 10 minutes, supernatants were discarded, and the pellet at the bottom of each tube resuspended with 1 ml DMEM. 50 µI of cell suspension was mixed with 50 µI ofTrypan Blue (Sigma cat. no. T8154) and incubated for 10 minutes. Total cell count was performed using the Countess-II Automated Cell Counter system (Life technologies cat.no.AMOAX10001).

Elastin and HA Staining
HOF (refer to Table 1) were seeded in 96-wells plates at a density of 10,000 ceHs/well in DMEM/10% FCS (LifeTech Holdings, cat. no.10270106). At approximately 80% confluence, medium was removed and remaining cells in the wells washed once with PBS. Cells were then cultured in DMEM/10% FCS (negative control) or RD-CLMSC-CM for 48 hours followed by immunocytochemical staining for HA (My BioSource, cat.no.MBS2025717) and elastin (LifeTech Holdings, cat.no.MA127129). Briefly, cells were washed with 1X PBS before fixation in cold methanol for 10 mins followed by blocking with 2.5% normal horse serum (Vector Laboratories cat.no.PK-7200) for 20 mins in a humid chamber. Cells were then incubated with primary HA antibodies (1:50) and elastin antibodies (1:50) for 2 hours in room temperature. They were washed with TBS (VWR Life Science cat.no.788) and 1% Tween solution (Sigma cat.no.P9416), followed by incubation with secondary antibodies (Vector Laboratories cat.no.PK-7200) for 15 mins. Washing was performed a further 3 times with lXTBS / 1% tween solution before incubation with ABC Reagent (Vector Laboratories cat.no.PK-7200) for 15 mins in room temperature. Finally, DAB (Dako cat.no.K3468) was added for colorimetric development. Harris Haematoxylin (Sigma cat.no.HHS16) was used for counterstaining. Images were taken with a bright field phase contrast microscope (Olympus) at 10X magnification. Expression of Elastin and Hyaluronic Acid were quantified with image J (National Institutes of Health, United States).

Results

Proliferative Effects of the Different CM vs DMEM/10% FCS DMEM/10% FCS (control) is commonly used for the expansion of mesenchymal cells such as HOF. Aged skin HDFs isolated from donors aged 60 and above were used instead of normal HDFs to amplify the difference in proliferative effects of RD-CLMSC-CM, H-CLMSC-CM, and FSF-CM. In comparison to DMEM/10% FCS (control), the highest increase in average total cell counts was seen in the RD-CLMSC-CM group (113% increase over control), followed by H-CLMSC-CM (112%), and FSF-CM {16%) (Figure 2). These results show that the proliferative factors in RD-CLMSC are as efficacious as those in H'-CLMSC-CM and demonstrate cross species efficacy without toxic effects on HDF.

Effect of DMEM/10% FCS vs RD-CLMSC-CM on the Expr,ession of HDF Elastin and HA
To compare the effects of RD-CLMSC-CM and DMEM/10% FCS (control) on the regulation of elastin and HA expression in HOF, and to assess if these effects were dependent on the age of the donors and the sites from which skin tissue were procured, HOF (n = 14) isolated from donors from 23 to 73 years in age and from multiple donor sites (forehead, eyelid, cheek, neck, tempile} were selected from a skin library for this set of experiments.The cells were subjected to 48 hours culture in either DIMEM/10% FCS or RD-CLMSC-CM before immunocytochemical staining for elastin and HA. Expression levels were quantified as optical densities using lmage J (National Institutes of Health, United States). RD­ CLMSC-CM upregulated the expression of elastin by 56% and HA by 83% in HOF as compared with DMEM/10% FCS (Figure 4). Overall, this set of experiments demonstrated significant upregulation of HOF elastin and hyaluronic acid expression.This effect appeared to be independent of the donor age or the donor site from which the skin was derived.

Figure 1
Steps in the isolation of Red Deer Umbilical Cord Lining Membrane. (1A) UC: Red Deer Umbilical Cord; (18) CL: Umbilical Cord Lining Membrane; G: Gelatinous tissue (equivalent to Wharton's jelly) within the umbilical cord. Side by side comparison of Umbilical Cord Lining Mesenchymal Stem Cells derived from Red Deer (lC) RD-CLMSC and Human (1□)H-CLMSC, maintained in subconfluent monolayers, show a similar morphology. Images were obtained with a phase-contrast microscope at 20x magnification.

Figure 2
Total cell counts were performed to compare the proliferative effects ofDMEM/10% FCS (Control),Human Foreskin Fibroblast conditioned media (FSF-CM), Human Cord Lining Mesenchymal Stem Cell Conditioned Media (H-CLMSC-CM), and Red Deer Cord Lining Mesenchymal Stem Cell Conditioned Media (RD-CLMSC-CM) on aged skin HDF(#asf74, #asf75, #asf76). Starting with the same seeding density and after 5 days incubation in the test medium, average total cell counts were calculated for each group. In comparison to the control, the highest increase in average total cell counts was seen in the RD-CLMSC-CM (113%), followed by H-CLMSC-CM group (112%), and then the FSF-CM group (16%).

Figure 3
Representative images of immunohistochemical staining of elastin and hyaluronic acid in HDF after culture in DMEM/10% FCS (control) vs RD-CLMSC-CM for 48 hours_ To compare the effects of DMEM/10% FCS and RD-CLMSC-CM on the expression of elastin and HA, HOF (n = 14) with varying donor profiles subject age from 23 to 73 years), skin from various sites (forehead, eyelid, cheek, neck, temple) were cultured in DMEM/10% FCS (control) and RD-CLMSC-CM for 48 hours before immunocytochemical staining for elastin and HA. Optical density was measured using lmage J. RD-CLMSC-CM upregulated HOF expression of elastin by 56% and hyaluronic acid by83% compared with HDF cultured in OMEM/10% FCS (control).

Discussion

As the active ingredient in a cosmeceutical product, RD­ CLMSC-CM appeared to increase skin turgor and elasticity in a prospective, randomized, double blinded, split face, placebo controlled clinical trial.49 The cosmeceutical cream (CALECIM® Professional Multi-Action Cream, CALECIM Cosmeceuticals, Singapore) contains 50% v/v RD-CLMSC-CM integrated into a proprietary transepidermal vehicle to allow the penetration of protein growth factors into the epidermis.The current study was conducted to elucidate the possible mechanisms of action of RD-CLMSC-CM on human skin.

Intrinsic skin aging results in multiple changes in the skin, including the decline in the levels of extracellular matrix (ECM) proteins including collagen,so.52 hyaluronic acid (HA),53•55 and elastin.56•57 The function of collagen in the skin is to provide firmness, whereas elastin fibers provide elasticity, and HA retains moisture by binding water molecules within the skin.ss.60

Reduction in the generation of theseECM proteins with skin aging results in poor elasticity seen as wrinkles and dermal atrophy from loss of moisture retention.61 To investigate if the significant improvements to skin elasticity were due to upregulation in elastin and HA expression, HOF isolated from different sites of the body from donors ages 23 to 73 years old were subjected to 48 hours culture in DMEM/10% FCS (control) and RD-CLMSC­ CM. Optical density measurements of immunocytochemistry staining revealed RD-CLMSC-CM upregulated the expression of elastin by 56% and HA by 83% in HOF as compared with control (Figure 3).

In humans, Transforming Growth Factor-131 (TGF-131) plays a central role in the expression of tropoelastin (TE), the soluble form of elastin.28 TGF-131 also stabilizes tropoelastin mRNA transcripts.29•30 Interestingly, a combination of TGF-131 and HA oligomers synergistically enhanced the levels of elastin in the extracellular matrix of cultured vascular smooth muscle cells.31 TGF-131 additionally reduced the degradation of elastin through reduction in the levels and activity of elastolytic proteases, including matrix metalloproteinase (MMP) -2 and -9.32Treatment of HDFs with various concentrations of TGF-131 or TGF-132 for 24 hours resulted in a dose-dependent elevation in the elastin mRNA steady-state levels, with a maximum 30 fold increase when dosed with 1 ng/ml.29

Insulin-like Growth Factor-1 (IGF-I) increases levels of tropoelastin mRNA and thus elastin production in neonatal aortic smooth muscle cells.47 Specificity Protein 1 (Sp1) promotes elastin transcription, whereas Sp3 suppresses Sp1- mediated enhancement of elastin transcription. IGF-I regulates elastin expression through the promotion of Sp1 binding to the elastin promoter and abrogation of Sp3 interaction with the elastin promoter.48 RD-AMSC-CM was positively identified with bFGF, IGFBP, IGFs, GM-CSF, PDGFs, VEGF, andTGF-!32.62 The upregulation of elastin expression can be postulated to be due to presence of TGF-l3s and IGFs found in RD-CLMSC-CM and all HOF demonstrated upregulation of elastin production after exposure to it.

In mammals, 3 Hyaluronic Acid Synthase (HAS) enzymes: HAS- 1, -2, and -3 synthesize HA chains of various lengths.63-65 Gene expression of HAS-1 and HAS-2 in the dermis and epidermis is differentially upregulated byTGF-l31.33•34 The mRNA expression of HAS-2 and HAS-3 can be stimulated by Fibroblast Growth Factor (FGF)-7, which activates keratinocyte migration and stimulates wound healing, leading to the accumulation of intermediate­ sized HA in culture medium and within keratinocytes.TGF-132 and Platelet Derived Growth Factor (PDGF)-88 have been shown to induce expression of HAS-2 as well as hyaluronan synthase by osteoblasts.66 We postulate that the significant upregulation of HA expression observed in all exposed HOF could be attributed to the presence ofTGF-l3s, PDGF-88, and FGF-7 present in RD­ CLMSC-CM.

The proliferative properties of RD-CLMSC-CM, H-CLMSC-CM, FSF-CM were investigated and compared with DMEM/10% FCS (control) by exposing aged HOF to these media over a period of 5 days (Figure 2). Slow proliferating aged HOF was used in this assay instead of the normal proliferating HOF to amplify the difference in the proliferative effects of the different CMs. FCS stimulates mammalian cellular growth in culture by providing essential components for cell proliferation and maintenance such as vitamins, hormones, transport proteins, trace elements, and mobility and growth factors.67.68 Compared to the FCS control, the average total cell counts in the RD­ CLMSC-CM group recorded the highest increase over control at 113%, followed closely by H-CLMSC-CM group at 112%; while the FSF-CM group recorded the lowest increase at 16% (Figure 2).The superior proliferative properties of CLMSC-CM (RD and H) over FSF-CM and the FBS control suggests the presence of other protein growth factors present in the CLMSC-CM that might be exerting a positive influence on HOF proliferation. RD antlerogenic derived MSCs conditioned medium (RD-AMSC­ CM) significantly enhanced the proliferation of HaCaTs and HDFs as compared with adipose derived mesenchymal stem cell conditioned medium (ASC-CM).62 RD genome was also identified to contain fibroblast growth factor-7 (FGF-7) gene. FGF-7 is known to play an important role in cellular proliferation and is a major paracrine effector of normal epithelial cell proliferation. (https://www.uniprot.org/uniprot/Q5DOX0).

VEGF significantly increased the proliferation capacity of HOF and human lung fibroblasts.24•25 HGF was shown to induce proliferation of human keratinocytes with the same potency as FGF-7.20 MSC used as a feeder layer to provide growth factors and essential components for the expansion of hemopoietic stem cells (HSCs). It appears that MSC induced proliferation of HSCs by the maintenance of HSCs in an undifferentiated state70 and also by reducing apoptosis.71 In addition to the expression of proliferative factors, CLMSC-CM (RD and H) may contain antiapoptotic and maintenance factors to retard cellular senescence and slow down growth arrest and reduce apoptosis. Overall, the net result would be higher total cell counts.

These experimental results demonstrate: 1) RD-CLMSC-CM is not toxic to HOF and does not induce cellular abnormalities; 2) Cross-species efficacy exists whereby protein cytokines from Red Deer exosomes can exert proliferative and pro-migratory responses in HOF; and 3) RD-CLMSC-CM can stimulate significant HOF production of HA and elastin. Taken together, these in vitro effects of RD-CLMSC-CM on HOF highlight the cellular basis to the clinical improvements in skin turgor reported by trial subjects after application of a cosmeceutical cream containing RD-CL-MSC-CM.