Isoforms of p63 in corneal stem cells cultured on human amniotic membrane

The p63 gene supports stem cell proliferation and regulation in epithelial cells. In this study, corneal epithelial cells were cultured on human amniotic membrane (HAM) and investigated for p63 and its isoform genes. Human limbal biopsies obtained from cadaveric donor eyes were cultivated on intact and denuded HAM. Transactivation (TA) specific domain was positive in the limbal cells cultured over denuded HAM and negative on others. TAp63α,β,γ isoforms are negative in all the limbal cells cultured on intact, denuded and limbal tissues but not in cornel epithelial tissue. p63α isoform is present in all except on denuded HAM. αβ sharing region is not expressed only in cornel epithelial tissue. γ isoform is expressed in all the samples. ΔNp63α region is present in cells cultured over the intact HAM whereas it is negative on the cells cultured over the denuded HAM. The other isoforms such as ΔNp63β and ΔNp63γ are negative in all samples. The limbal cells cultured over the intact HAM were able to maintain high proliferative potential when compared to denuded HAM. Thus, p63 isoforms plays a biological function to retain the proliferative capacity of corneal epithelial cells and maintains the stemness when cultured on intact HAM.     

Culture of human amniotic fluid stem cells in 3D collagen matrix

Most of the researchers attribute amniotic fluid stem cells (AF SCs) to mesenchymal stem cells (MSCs). However, AF SCs express both mesenchymal and epithelial markers, which distinguishes them from postnatal MSCs. Cultivation in the three-dimensional (3D) matrix provides a different look at the nature of the cells. We showed that in 3D collagen gel AF SCs form epithelial structures (tubules and cysts). The active contraction of the gel during the first days of cultivation, which is characteristic of mesenchymal cells, does not occur. Electron microscopic study showed that adherent junctions typical to epithelial cells are formed between AF SCs. On the other hand, during culturing in the gel AF SCs continue to express MSCs markers. Thus, AF SCs may be not true mesenchymal cells because they can display properties of epithelial cells. Perhaps these cells undergo epithelial-mesenchymal transition, a process which actively takes place during embryogenesis.     

Comparison of characteristics of cultured limbal cells on denuded amniotic membrane and fresh conjunctival, limbal and corneal tissues

This study aimed to evaluate proposed molecular markers related to eye limbal stem cells (SC) and to identify novel associated genes. The expression of a set of genes potentially involved in stemness was assessed in freshly prepared limbal, corneal and conjunctival tissues. PAX6, AC133, K12 and OCT4 were detected in all the tissues and p63(+)/K3(-)/K12(+)/Nodal(+)/Cx43(+) were expressed in conjunctival, p63(-)/K3(+)/K12(+)/Nodal(-)/Cx43(+) in corneal, and p63(+)/K3(-)/K12(-)/Nodal(-)/Cx43(-) in limbal tissues. Limbal explants were cultured on human amniotic membrane for 21 days. The cells expressed p63 but not K3, K12, Nodal and Cx43, however, the expression of K3, K12 and Cx43 was detected, and p63 and the high BrdU-labeling index decreased with more culture. Ultrastructure analysis of the cultured cells showed typically immature organization of intracellular organelles and architecture. Our data suggest that limbal, corneal and conjunctival tissues are heterogeneous with some progenitors. Also, the expression of traditional SC markers may not be a reliable indicator of limbal SC and there is an increasing need to determine factor(s) involved in their stemness.     

The culture and transplantation of human limbal stem cells

The cornea is the clear front of the eye and its surface is composed of an epithelium. This is renewed by stem cells located at the limbus, which encircles the periphery of the cornea. These limbal stem cells become lost or deficient in the blinding disease of limbal stem cell deficiency. In this review article, we discuss the historical perspective in managing limbal stem cell deficiency as well as describing the more contemporary treatment options, and in particular the culture and transplantation of human limbal stem cells. This treatment was first proposed 13 years ago and many case series have been presented to date showing promising outcomes of this technique. However, challenges still remain in treating the debilitating disease of limbal stem cell deficiency. Here we discuss some of the questions, which remain to be answered in this field. J. Cell. Physiol. 225: 15–19, 2010. © 2010 Wiley‐Liss, Inc.     

Serum eye drops,amniotic membrane and limbal epithelial stem cells—tools in the treatment of ocular surface disease

The advent of serum eye drops, amniotic membrane and limbal stem cell grafts, have transformed the treatment of diseases which result in ocular surface failure. This article provides an overview of ocular surface anatomy and failure, and how the use of serum eye drops, amniotic membrane and limbal epithelial stem cell transplantation has influenced ophthalmological practice both historically and in recent years. This review focuses on the rationale for the use of these emerging tools, how they are prepared, clinical applications, limitations, and speculates on future directions.     

Therapeutic use of limbal stem cells

Stem cells are defined as relatively undifferentiated cells that have the capacity to generate more differentiated daughter cells. Limbal stem cells are responsible for epithelial tissue repair and regeneration throughout the life. Limbal stem cells have been localized to the Palisades of Vogt in the limbal region. Limbal stem cells have a higher proliferative potential compared to the cells of peripheral and central cornea. Limbal stem cells have the capacity to maintain normal corneal homeostasis. However, in some pathological states, such as chemical and thermal burns, Stevens-Johnson syndrome, and ocular pemphigoid limbal stem cells fail to maintain the corneal epithelial integrity. In such situations, limbal stem cell transplantation has been required as a therapeutic option. In unilateral disorders, the usual source of stem cells is the contralateral eyes, but if the disease is bilateral stem cell allografts have to be dissected from family members or cadaver eyes. The advent of ex vivo expansion of limbal stem cells from a small biopsy specimen has reduced the risk of limbal deficiency in the donor eye. Concomitant immunosuppressive therapy promotes donor-derived epithelial cell viability, but some evidences suggest that donor-derived epithelial stem cell viability is not sustained indefinitely. Thus, long-term follow-up studies are required to ascertain whether donor limbal stem cell survival or promotion of recolonization by resident recipient stem cells occurs in restored recipient epithelium. However, this is not an easy task since a definitive limbal stem cell marker has not been identified yet. This review will discuss the therapeutic usage of limbal stem cells in the corneal epithelial disorders.     

Ex vivo characteristics of human amniotic membrane-derived stem cells

Cells were isolated from four human amniotic membranes, and their biological characteristics analyzed during ex vivo expansion. Morphologically homogenous populations of fibroblast-like cells were obtained from the second or third passage. Under the appropriate culture conditions, these human amniotic membrane-derived mesenchymal cells (HAM) were shown to differentiate into adipocytes, osteocytes, chondrocytes and neuronal cells, as visualized by Oil Red O, von Kossa, alcian blue, anti-Neu N, and anti-Gal C antibody staining, respectively. Immunophenotype analysis of HAM cells revealed the presence of antigens for SSEA-3, SSEA-4, collagen type-I, -II, -III, -IV, -XII, fibronectin, alpha-SMA, vimentin, desmin, cytokeratin18 (CK18), HCAM-1, fibroblast surface protein, and human leukocyte antigen (HLA) ABC. ICAM-1 protein was weakly detectable, and proteins of TRA-1-60, VCAM-1, von Willebrand factor, PECAM-1, and HLA DR were not detected. HAM cells reached senescence after 14.5+/-0.9 passages, over a period of 146.8+/-8.9 days, and underwent an average of 36.9 4.7 population doublings. RT-PCR analysis showed that all four HAM cell lines consistently expressed genes of Oct-4, Rex-1, SCF, NCAM, nestin, BMP-4, GATA-4, HNF-4alpha, vimentin, and CK18, regardless of the passage number. The genes of Brachyury, FGF-5, Pax-6, and BMP2 were never expressed. Strikingly, alpha-fetoprotein (alphaFP), HLA ABC, and HLA DR genes were expressed in an earlier passage but not expressed in later passages. Telomerase activity of two HAM lines was discernable upon the third passage. These observations strongly suggest that HAM might be immune-privileged and, thus, advantageous as therapeutic cells.     

Critical appraisal of ex vivo expansion of human limbal epithelial stem cells

The stem cells (SCs) of the corneal epithelium located in the limbal basal layer are the ultimate source to maintain corneal epithelial homeostasis. Like other adult tissue-specific SCs, self renewal and fate decision of limbal SCs are regulated by a specialized in vivo microenvironment, termed "niche". Loss of limbal SCs or dysfunction of the limbal niche renders corneas with a unique clinical disease labeled limbal stem cell deficiency (LSCD). Besides transplantation of autologous or allogeneic limbal SCs or amniotic membrane, a new strategy of treating LSCD is to transplant a bio-engineered graft by expanding limbal SCs ex vivo. Herein, we conduct a critical appraisal of six protocols that have successfully been practiced in treating human patients with LSCD, and identify issues whether niche regulation has been disrupted or maintained during isolation and expansion. Consequently, we propose a future direction that may circumvent the potential pitfalls existing in these conventional protocols by preserving the interaction between limbal SCs and their native niche cells during isolation and expansion. Such an approach may one day help realize considerable promise held by adult SCs in treating a number of diseases.     

Towards xeno-free cultures of human limbal stem cells for ocular surface reconstruction

Isolated limbal epithelial stem cells (LESCs) were cultured with or without a 3T3 murine fibroblast feeder-layer (FL) in 4 different culture media on culture plates or on denuded human amniotic membrane (AM) support and fibrin gel support: (1) control medium supplemented with fetal bovine serum; (2) control medium supplemented with the synthetic serum “XerumFree? XF205” (XF); (3) CnT-20 medium supplemented with “XerumFree? XF205” (CnT-XF) and (4) CnT-20 medium supplemented with human AB serum (CnT-AB). The three xenogeneic media were compared to standard condition (control + FL) and parameters assessed included cell morphology, proliferative potential, number of passages, assessment of clonogenic and abortive colonies, life span, ?Np63α expression and epithelial morphology on AM. During serial cultivation of LESCs, most of the tested xeno-free media supported similar numbers of cell passages, total colony number, cumulative cell doublings (CCD) rates and expression of ?Np63α compared to control. The conditions cultivated with a FL showed a non-statistically significant higher number of cell passages and CCD rates before senescence when compared to the same conditions cultured without FL. Except for the control medium, only XF medium enabled the growth of cells on AM. The expression of ?Np63α was comparable in all the cultures grown onto AM, when compared to the controls on fibrin gel. In conclusion, the xeno-free media enabled LESC culture both on plastic and on denuded human AM. Despite the analyses were carried out in a statistically low number of samples and need re-assessment in a larger cohort, our results suggest that the production of a completely xeno-free LESC graft could be beneficial for future clinical applications.     

Differentiation of human amniotic membrane cells into osteoblasts in vitro

OBJECTIVE: Pluripotent stem cells may be exist in the human amniotic membrane (HAM). The present article was aimed at establishing HAM cell lines and investigating their differentiation into osteoblasts in vitro. METHODS: HAM cell lines were established using routine cell culture techniques and expanded in vitro. RESULTS: HAM cells were propagated from 45 out of 50 specimens (90%) and all were maintained normal karyotypes in vitro. Alkaline phosphatase (ALP) activity and alizarin red S stain were positive for 12 out of 22 HAM cell lines (54.5%). The 22 HAM cell lines were selected at random. CONCLUSIONS: This study demonstrates that this isolation method II was more effective for establishing cell lines which differentiate into osteoblast rather than method I.     

Differentiation of human amniotic membrane cells into osteoblasts in vitro

OBJECTIVE: Pluripotent stem cells may be exist in the human amniotic membrane (HAM). The present article was aimed at establishing HAM cell lines and investigating their differentiation into osteoblasts in vitro. METHODS: HAM cell lineswere established using routine cell culture techniques and expanded in vitro. RESULTS: HAM cells were propagated from 45 out of 50 specimens (90%) and all were maintained normal karyotypes in vitro. Alkaline phosphatase (ALP) activity and alizarin red S stain were positive for 12 out of 22 HAM cell lines (54.5%). The 22 HAM cell lines were selectedat random. CONCLUSIONS: This study demonstrates that this isolation method II was more effective for establishing cell lines which differentiate into osteoblast rather than method I.     

The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy

The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140]     

Effects of human amniotic membrane grafts combined with marrow mesenchymal stem cells on healing of full-thickness skin defects in rabbits

We have investigated the wound-healing effects of mesenchymal stem cells (MSCs) in combination with human amniotic membrane (HAM) when grafted into full-thickness skin defects of rabbits. Five defects in each of four groups were respectively treated with HAM loaded with autologous MSCs (group A), HAM loaded with allologous MSCs (group B), HAM with injected autologous MSCs (group C), and HAM with injected allologous MSCs (group D). The size of the wounds was calculated for each group at 7, 12, and 15 days after grafting. The wounds were subsequently harvested at 25 days after grafting. Sections stained with hematoxylin and eosin were used to determine the quality of wound healing, as based on the characteristics and amount of granulated tissue in the epidermal and dermal layers. Groups A and B showed the most pronounced effect on wound closure, with statistically significant improvement in wound healing being seen on post-operative days 7, 12, and 15. Although a slight trend toward improved wound healing was seen in group A compared with group B, no statistically significant difference was found at any time point between the two groups. Histological examination of healed wounds from groups A and B showed a thin epidermis with mature differentiation and collagen bundle deposition plus recovered skin appendages in the dermal layer. In contrast, groups C and D showed thickened epidermis with immature epithelial cells and increased fibroblast proliferation with only partially recovered skin appendages in the dermal layer. Thus, the graft of HAM loaded with MSCs played an effective role during the healing of skin defects in rabbits, with no significant difference being observed in wound healing between autologous and allologous MSC transplantation. This study was supported by research funds from Dong-A University.     

人羊膜间充质细胞分离培养及其干细胞特性研究

目的:研究人羊膜间充质细胞(Humanamnioticmesenchymalcells,HAMCs)的分离、培养及其干细胞特性,为羊膜间充质细胞在再生医学的潜在应用奠定实验基础。方法:无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,经胰酶胶原酶序贯消化,DMEM/F12培养,倒置显微镜下观察其形态,MTT法检测其生长规律,免疫荧光的方法对细胞进行鉴定,定向诱导方法检测细胞的多向分化潜能。结果:来源于羊膜的间充质细胞,细胞免疫荧光显示SSEA-4,OCT-4阳性,具有很强的增殖能力,并且具有一定的多向分化能力,在特定条件下可分化为脂肪细胞和成骨细胞;结论:羊膜间充质细胞能够在体外分离、培养、扩增,并且具有干细胞特性。羊膜间充质细胞在再生医学和组织工程应用有很好的前景。     

人羊膜间充质细胞分离培养及其干细胞特性研究

目的：研究人羊膜间充质细胞（Humanamnioticmesenchymalcells,HAMCs）的分离、培养及其干细胞特性,为羊膜间充质细胞在再生医学的潜在应用奠定实验基础。方法：无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,经胰酶胶原酶序贯消化,DMEM/F12培养,倒置显微镜下观察其形态,MTT法检测其生长规律,免疫荧光的方法对细胞进行鉴定,定向诱导方法检测细胞的多向分化潜能。结果：来源于羊膜的间充质细胞,细胞免疫荧光显示SSEA-4,OCT-4阳性,具有很强的增殖能力,并且具有一定的多向分化能力,在特定条件下可分化为脂肪细胞和成骨细胞;结论：羊膜间充质细胞能够在体外分离、培养、扩增,并且具有干细胞特性。羊膜间充质细胞在再生医学和组织工程应用有很好的前景。     

Human amniotic membrane as an alternative source of stem cells for regenerative medicine

The human amniotic membrane (HAM) is a highly abundant and readily available tissue. This amniotic tissue has considerable advantageous characteristics to be considered as an attractive material in the field of regenerative medicine. It has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Since it is discarded post-partum it may be useful for regenerative medicine and cell therapy. Amniotic membranes have already been used extensively as biologic dressings in ophthalmic, abdominal and plastic surgery. HAM contains two cell types, from different embryological origins, which display some characteristic properties of stem cells. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. Both populations have similar immunophenotype and multipotential for in vitro differentiation into the major mesodermal lineages, however they differ in cell yield. Therefore, HAM has been proposed as a good candidate to be used in cell therapy or regenerative medicine to treat damaged or diseased tissues.