Research Article: Human Wharton’s Jelly Mesenchymal Stem Cells Plasticity Augments Scar-Free Skin Wound Healing with Hair Growth

Date Published: April 15, 2014

Publisher: Public Library of Science

Author(s): Vikram Sabapathy, Balasubramanian Sundaram, Sreelakshmi VM, Pratheesh Mankuzhy, Sanjay Kumar, Aditya B. Pant.


Human mesenchymal stem cells (MSCs) are a promising candidate for cell-based transplantation and regenerative medicine therapies. Thus in the present study Wharton’s Jelly Mesenchymal Stem Cells (WJ-MSCs) have been derived from extra embryonic umbilical cord matrix following removal of both arteries and vein. Also, to overcome the clinical limitations posed by fetal bovine serum (FBS) supplementation because of xenogeneic origin of FBS, usual FBS cell culture supplement has been replaced with human platelet lysate (HPL). Apart from general characteristic features of bone marrow-derived MSCs, wharton jelly-derived MSCs have the ability to maintain phenotypic attributes, cell growth kinetics, cell cycle pattern, in vitro multilineage differentiation plasticity, apoptotic pattern, normal karyotype-like intrinsic mesenchymal stem cell properties in long-term in vitro cultures. Moreover, the WJ-MSCs exhibited the in vitro multilineage differentiation capacity by giving rise to differentiated cells of not only mesodermal lineage but also to the cells of ectodermal and endodermal lineage. Also, WJ-MSC did not present any aberrant cell state upon in vivo transplantation in SCID mice and in vitro soft agar assays. The immunomodulatory potential assessed by gene expression levels of immunomodulatory factors upon exposure to inflammatory cytokines in the fetal WJ-MSCs was relatively higher compared to adult bone marrow-derived MSCs. WJ-MSCs seeded on decellularized amniotic membrane scaffold transplantation on the skin injury of SCID mice model demonstrates that combination of WJ-MSCs and decellularized amniotic membrane scaffold exhibited significantly better wound-healing capabilities, having reduced scar formation with hair growth and improved biomechanical properties of regenerated skin compared to WJ-MSCs alone. Further, our experimental data indicate that indocyanin green (ICG) at optimal concentration can be resourcefully used for labeling of stem cells and in vivo tracking by near infrared fluorescence non-invasive live cell imaging of labelled transplanted cells, thus proving its utility for therapeutic applications.

Partial Text

Mesenchymal stromal cells (MSCs) are a pluripotent class of stem cells that has the ability to self-renew and differentiate into multiple cell lineages. Friedenstein et al first isolated and recognized the multilineage differentiation ability of mesenchymal stromal cell [1]. The mesenchymal stromal cells can be broadly classified into two categories; MSCs derived from adult tissues such as bone marrow, adipose tissue [2] and fetal/perinatal tissues derived such as placenta [3], umbilical cord wharton’s jelly [4], amniotic membrane etc.[5]. Adult MSCs are the most commonly used MSCs but the proliferative capacity of adult MSCs are very limited, making it very difficult to scale up these adult MSCs for therapeutic applications [6]. Hence, alternate source of mesenchymal stromal cells is required for clinical application. The Mesenchymal stromal cells from extra embryonic tissues is an ideal choice for mesenchymal stem cells, as it can overcome the proliferative limitation posed by adult MSCs. Further, fetal MSCs has proliferation capacity, ease of scalability, differentiation plasticity and exhibits some of the gene expression characteristic features of embryonic stem cells without any tumorigenicity. Additionally, the immunomodulatory potential of fetal MSCs renders them as an attractive choice for regenerative medical applications [7]. In 1656 Thomas Wharton first reported the description of human umbilical chord [8]. McElreavey et al., [9] in 1991 first isolated the mesenchymal stromal cells from wharton’s jelly portion of the umbilical cord. Previous studies indicate that WJ-MSCs can be used for broad range of applications such as neurological disorders [10], kidney injury [11], lung injury [12], orthopedic injury [13], liver injury [14], cancer therapy [15]. Recent advances suggest that WJ-MSCs reinforced with microparticles [16] and scaffolds [17] can be effectively used for variety of clinical applications. Auxiliary reports suggest that paracrine factors secreted by the MSCs play a very vital role in therapeutic, immunomodulatory and tissue regeneration capabilities of MSCs [18].

Use of FBS as a media supplement has lot of disadvantages for clinical applications. Use of platelet lysate as an alternative in vitro cell culture supplement might lower the limitations, posed by the use of fetal bovine serum (FBS). In this study the human WJ-MSCs was successfully isolated and in vitro culture expanded by using 5% HPL supplement in α-MEM Media. The calculated population doubling time was around 10 hours. The isolated human WJ-MSCs were subjected to primary characterization analysis such as flow cytometry and differentiation analysis. The characteristics property exhibited by the WJ-MSCs were similar to that of adult bone marrow derived MSCs. Previous studies have indicated that human WJ-MSCs exhibits similar phenotypic characteristics, surface marker expression, differentiation ability, immunomodulatory effects and paracrine factors similar to that of adult bone marrow MSCs [33]. Nonetheless, Fetal WJ-MSCs exhibited high proliferation capacity compared to adult BM-MSCs thus can be scaled up easily for regenerative medical applications. WJ-MSCs exhibited the capacity to transgress across the trans-lineage barrier and able to efficiently differentiate into ectoderm (neural cells), mesoderm (adipocyte, osteocyte, chondrocyte, smooth muscles etc.) and endoderm (Pancreatic progenitors). Additionally, isolated human WJ-MSCs were subjected to secondary characterization studies such as cell cycle analysis, apoptosis analysis, cytogenetics and in vitro tumorigenesis analysis. The overall results suggest that isolated cells were very healthy and did not form any aberrant cell types upon extended passaging. The MSCs isolated from extra-embryonic tissues such as umbilical cord has pluripotency marker expression level some were midway between embryonic and adult cells [6]. Previous studies have indicated the presence of higher expression of pluripotency marker such as Nanog, OCT4, Tra-1-81 etc., in the cells isolated from the extra embryonic tissues [34]. According to our data, comparative real-time PCR analysis between the adult bone marrow derived MSCs and WJ-MSCs indicate the presence of considerable levels of Nanog and Sox2 expression. The presence of high level of pluripotency marker in the fetal MSCs compared to adult BM-MSCs might play a vital role in improved proliferation and differentiation capabilities. Initial reports on immunoregulatory property of adult MSCs were demonstrated just around a decade back [35], [36]. MSCs have the ability to modulate both innate and adaptive immune response by complex interactions with T cells, B cells, NK cells, dendritic cells, macrophages, neutrophils, toll like receptors etc., [37]. When the MSCs were subjected to proinflammatory cytokines such as IL-1β, TNFα and INFα-2b, there was a differential expression level of immunomodulatory molecules such as TGFβ1, IDO, PGE2 and TSG6. Over fetal WJMSCs exhibited comparatively elevated expression of immunomodulatory molecules when compared to adult bone marrow MSCs. The WJ-MSCs express lower levels of HLA-I thus provokes no immune response from the host. Our data is consistent with previously reported result that, WJMSCs exhibits better immunomodulatory properties compared to adult BM derived MSCs [38]. Nonetheless, still the complex mechanism of immunomodulation of MSCs remains shrouded in mystery. Wound healing is a convoluted process involving coordinated interplay between cells, growth factors and extracellular proteins. The MSCs plays a vital role by secreting important paracrine factors that helps coordinating the wound healing process. The prior reports on various animal models and clinical trials indicate MSCs play a beneficial role in augmenting the wound healing process [39]. In this study, the data suggest that the grafting of the WJ-MSCs seeded decellularized amniotic membrane exhibited better wound healing capabilities compared to injection of MSCs alone. The histological study of regenerated wound tissue two week post injury suggest that glandular tissues were better developed and organized in membrane grafted tissue compared to control or plain WJ-MSCs injected animal model. Further, one of our main goals was to successfully track the transplanted cell under in vivo conditions. ICG is FDA approved dye with an absorbance and emission peak at 780 nm and 830 nm respectively [40]. It is approved for clinical applications such as cardiac perfusion etc. In this study, we have optimized the protocol for efficient labeling of the cells. The ICG concentration of 0.2 mg/ml was successfully employed for both cell labeling as well as in vivo imaging. Moreover, previous reports have indicated that ICG greater than 0.5 mg/ml might decreases the viability of the cells. The ICG labeled cells seeded on decellularized amniotic membrane was grafted onto the area of dermal injury. The labeled cells were tracked via IVIS imaging system for almost 10 days. Thus, non-toxic ICG labeling can be successfully extrapolated for clinical applications to track the transplanted cells. Mechanical characterization data of the regenerated tissue supplemented our finding that combination of WJ-MSCs/scaffold is suitable for wound healing. The interpretation of modulus of toughness, ultimate tensile strength, young’s modulus and stiffness provides sufficient evidence that the wound healing capabilities of WJMSCs/Scaffold was significantly better compared to control. By taking account of only the mechanical parameters, there was no significant difference between WJ-MSCs and WJ-MSCs/Scaffold transplanted groups. However, Ultimate tensile strength, young’s modulus and stiffness values of WJ-MSCs/Scaffold transplanted group were more compared to only WJ-MSCs injected group.

Human platelet lysate was used to successfully expand the isolated WJ-MSCs from human wharton jelly tissue of umbilical cord. The primary and secondary characterization of isolated WJ-MSCs was carried to ascertain the phenotypic as well as genotypic features of the MSCs. The fetal MSCs exhibited greater stemness compared to gold standard adult bone marrow derived MSCs. Moreover, the higher immunomodulatory factors were secreted by the extra embryonic tissue derived MSCs compared to adult MSCs under proinflammatory conditions. SCID mice skin injury model suggest that combination of natural decellularised amniotic membrane scaffold along with WJMSCs can be effectively used to treat the skin injury. Lastly, the data indicate that ICG can be used as a reliable source for tracking of the cells during in vivo applications.