Francisco J. de Toro

Multilineage differentiation potential of cells isolated from the human amniotic membrane

The human amniotic membrane (HAM) contains two cell types from different embryological origins. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. In this study, we localized, isolated, quantified and phenotypically characterized HAM‐derived cells and analysed their in vitro differentiation potential towards mesodermal cell lineages. Human amnion‐derived cells were isolated and characterized by flow cytometry. Immunohistochemistry and quantitative real‐time reverse transcription‐polymerase chain reaction studies were performed for the analysis of multipotentiality. Immunophenotypic characterization of both cell types demonstrated the presence of the common, well‐defined human mesenchymal stem cell (MSC) markers (CD90, CD44, CD73, CD166, CD105, CD29), as well as the embryonic stem‐cell markers SSEA‐4 and STRO‐1. Phenotypes of both cell populations were maintained from passages P0 to P9. The assessment of multilineage potential demonstrated that the hAMSCs showed greater adipogenic and chondrogenic potential. Both populations had the ability to retain their capacity for differentiation during culture passages from P0 to P4. Our data demonstrate the successful localization and isolation of hAMSCs and hAECs from the HAM. Both cell populations possessed similar immunophenotype. However, they differed in cell yield and multipotential for differentiation into the major mesodermal lineages. Our functional differentiation studies demonstrated that hAMSCs possess a much greater mesodermal differentiation capacity than hAECs. These considerations will be important for use of these cells for cell therapy. J. Cell. Biochem. 111: 846–857, 2010.

Differentiation of synovial CD-105(+) human mesenchymal stem cells into chondrocyte-like cells through spheroid formation.

Mesenchymal stem cells (MSCs) have the capacity to differentiate into several cell lineages, some of which can generate bone, cartilage, or adipose tissue. The presence of MSCs in the synovial membrane was recently reported. Data from comparative studies of MSCs derived from various mesenchymal tissues suggest that MSCs from synovial membranes have a superior chondrogenesis capacity. Previous chondrogenic differentiation studies have used the total population of MSCs, including cells with several MSC markers, such as CD44, CD90, CD105, or CD73. However the chondrogenic capacity of an individual population of MSCs has not been examined. Our aim was to study the chondrogenic capacity of the cellular MSC subset, CD105+, derived from synovial membrane tissues of patients with osteoarthritis (OA) and normal donors. The tissues were digested with a cocktail of collagenase/dispase and the isolated MSCs were seeded into plates. The subpopulation of CD105+‐MSCs was separated using a magnetic separator. The MSCs were then differentiated towards chondrocyte‐like cells using a specific medium to promote spheroid formation. Spheroids were collected after 14, 28, and 46 days in chondrogenic medium and stained with hematoxylin, eosin, Safranin O or Alcian blue to evaluate the extracellular matrix. Immunohistochemistry was performed to study collagen types I (COLI) and II (COLII) and aggrecan expression. Phenotypic characterization of the isolated CD105+‐MSCs shows that these cells are also positive for CD90 and CD44, but negatives for CD34 and CD45. In addition, this cellular subset expressed Sox‐9. Spheroids appeared after 7 days in culture in the presence of chondrogenic medium. Our studies show no differences between MSCs obtained from OA and normal synovial membranes during chondrogenesis. The morphological analysis of spheroids revealed characteristics typical of chondrocyte cells. The intensity of Safranin O, Alcian blue and aggrecan staining was positive and constant throughout the culture period. However, the intensity of COL2 staining was higher at 28 days (84.29 ± 0.1 U) than at 46 days (61.28 ± 01 U), while COL1 staining was not detected in any samples analyzed. These results were confirmed by reverse transcriptase‐polymerase chain reaction assays. We conclude that the cellular subset of CD105+‐MSCs has chondrogenic capacity. The study also show the similar chondrogenic capacity of CD105+‐MSCs cultured from normal and OA synovial membranes. J. Cell. Biochem. 108: 145–155, 2009.

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.

Quantification of Cells Expressing Mesenchymal Stem Cell Markers in Healthy and Osteoarthritic Synovial Membranes

Objective. To quantify cells expressing mesenchymal stem cell (MSC) markers in synovial membranes from human osteoarthritic (OA) and healthy joints. Methods. Synovial membranes from OA and healthy joints were digested with collagenase and the isolated cells were cultured. Synovial membrane-derived cells were phenotypically characterized for differentiation experiments using flow cytometry to detect the expression of mesenchymal markers (CD29, CD44, CD73, CD90, CD105, CD117, CD166, and STRO-1) and hematopoietic markers (CD34 and CD45). Chondrogenesis was assessed by staining for proteoglycans and collagen type II, adipogenesis by using a stain for lipids, and osteogenesis by detecting calcium deposits. Coexpression of CD44, CD73, CD90, and CD105 was determined using immunofluorescence. Results. Cells expressing MSC markers were diffusely distributed in OA synovial membranes; in healthy synovial membrane these cells were localized in the subintimal zone. More numerous MSC markers in OA synovial membranes were observed in cells also expressing the CD90 antigen. FACS analysis showed that more than 90% of OA synovial membrane-derived cells were positive for CD44, CD73, and CD90, and negative for CD34 and CD45. OA synovial membrane-derived cells were also positive for CD29 (85.23%), CD117 (72.35%), CD105 (45.5%), and STRO-1 (49.46%). Micropellet analyses showed that the culture of cells with transforming growth factor-ß3 stimulated proteoglycan and collagen type II synthesis. Conclusion. Synovial membranes from patients with OA contain more cells positive for CD44, CD90, and CD105 antigens than those from joints with undamaged cartilage.

Analysis of the Chondrogenic Potential and Secretome of Mesenchymal Stem Cells Derived from Human Umbilical Cord Stroma

Mesenchymal stem cells (MSCs) from umbilical cord stroma were isolated by plastic adherence and characterized by flow cytometry, looking for cells positive for OCT3/4 and SSEA-4 as well as the classic MSC markers CD44, CD73, CD90, Ki67, CD105, and CD106 and negative for CD34 and CD45. Quantitative reverse transcriptase-polymerase chain reaction analysis of the genes ALP, MEF2C, MyoD, LPL, FAB4, and AMP, characteristic for the differentiated lineages, were used to evaluate early and late differentiation of 3 germ lines. Direct chondrogenic differentiation was achieved through spheroid formation by MSCs in a chondrogenic medium and the presence of chondrogenic markers at 4, 7, 14, 28, and 46 days of culture was tested. Immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction analyses were utilized to assess the expression of collagen type I, collagen type II, and collagen type X throughout the time studied. We found expression of all the markers as early as 4 days of chondrogenic differentiation culture, with their expression increasing with time, except for collagen type I, which decreased in expression in the formed spheroids after 4 days of differentiation. The signaling role of Wnt during chondrogenic differentiation was studied by western blot. We observed that β-catenin expression decreased during the chondrogenic process. Further, a secretome study to validate our model of differentiation in vitro was performed on spheroids formed during the chondrogenesis process. Our results indicate the multipotential capacity of this source of human cells; their chondrogenic capacity could be useful for future cell therapy in articular diseases.

Xeno‐implantation of pig chondrocytes into rabbit to treat localized articular cartilage defects: an animal model

Articular cartilage has only a limited ability to regenerate. The transplantation of autologous chondrocytes is currently used to treat focal defects in human articular cartilage, although use of organs, tissues, or cells from different species is being investigated as an alternative treatment. The object of this study was to use xeno‐transplantation of cultured pig chondrocytes for the repair of rabbit chondral defects, and to analyze the significance of tissue rejection in this animal model. Partial chondral defects, including removal of cartilage tissue and a part of the subchondral bone, were created in the lateral femoral condyles of 30 adult New Zealand White rabbits. A periosteal flap was sutured to the native cartilage with the cambium layer facing the defect. As a control, culture medium was injected into the defect void of one group of rabbits while in a treatment group, chondrocytes, isolated from normal femoral pig cartilage, were injected into the defect void. All rabbits were killed by 24 weeks. Macroscopic changes of the cartilage were analyzed using Mankins score. The distal femoral portion was studied histologically using hematoxylin and eosin, alcian blue, toluidine blue, and Masons trichrome. Pig cells and pig genetic material were detected in the neo‐synthesized tissue by immunohistochemical detection of SLA‐II‐DQ and polymerase chain reaction analysis of the gene SLA‐II‐DQB. The synovial membrane was studied histologically by hematoxylin and eosin staining. In the control group, on average, less than 25 percent of the chondral defect was filled. The repair tissue had an irregular surface with few cells similar to chondrocytes or fibroblasts and a minimal formation of extracellular matrix. In the treatment group, the chondral defect was approximately 90 percent filled with good integration between the neo‐synthesized cartilage and the native cartilage. The repair tissue had a smooth surface with cells similar to chondrocytes and a hyaline‐like extracellular matrix. The neo‐synthesized cartilage was morphologically similar to hyaline cartilage. Importantly, there were no signs of graft‐vs.‐host rejections or infiltration by immune cells. In the neo‐synthesized tissue, pig genetic material was detected in 27 ± 5 percent of all cells. These cells containing pig genetic material were distributed throughout the neo‐synthesized cartilage. We conclude that the xeno‐transplantation of chondrocytes could be an alternative method for the repair of articular cartilage defects.

Proteome Analysis During Chondrocyte Differentiation in a New Chondrogenesis Model Using Human Umbilical Cord Stroma Mesenchymal Stem Cells

Umbilical cord stroma mesenchymal stem cells were differentiated toward chondrocyte-like cells using a new in vitro model that consists of the random formation of spheroids in a medium supplemented with fetal bovine serum on a nonadherent surface. The medium was changed after 2 days to one specific for the induction of chondrocyte differentiation. We assessed this model using reverse transcriptase-polymerase chain reaction, flow cytometry, immunohistochemistry, and secretome analyses. The purpose of this study was to determine which proteins were differentially expressed during chondrogenesis. Differential gel electrophoresis analysis was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry protein identification. A total of 97 spots were modulated during the chondrogenesis process, 54 of these spots were identified as 39 different proteins and 15 were isoforms. Of the 39 different proteins identified 15 were down-regulated, 21 were up-regulated, and 3 were up- and down-regulated during the chondrogenesis process. Using Pathway Studio 7.0 software, our results showed that the major cell functions modulated during chondrogenesis were cellular differentiation, proliferation, and migration. Five proteins involved in cartilage extracellular matrix metabolism found during the differential gel electrophoresis study were confirmed using Western blot. The results indicate that our in vitro chondrogenesis model is an efficient and rapid technique for obtaining cells similar to chondrocytes that express proteins characteristic of the cartilage extracellular matrix. These chondrocyte-like cells could prove useful for future cell therapy treatment of cartilage pathologies.

Pig chondrocyte xenoimplants for human chondral defect repair: an in vitro model

The objective of this study was to evaluate the use of cultured porcine chondrocyte xenotransplantation for the repair of human chondral defects. Two‐millimeter‐diameter defects were drilled into explants of femoral cartilage from healthy adult donors. No cells were implanted in the chondral defects of the control group, while pig chondrocytes from normal femoral cartilage were deposited into the treated chondral defects. Cartilage explants were cultured for 4, 8, and 12 weeks. Tissue sections were processed for standard histologic staining and immunostaining with monoclonal antibodies against types I and II collagen, chondroitin‐4‐sulfate, chondroitin‐6‐sulfate, keratan sulfate, and integrin subunit β1. The porcine origin of chondrocytes was confirmed using a specific pig monoclonal anti‐CD46. Repair was only observed in the cell‐treated defects. Mono‐ or bilayers of cells were detected after 4 culture weeks on the bottom of the defects, while after 8–12 weeks a repair tissue filled near 30–40 percent of the defect. At 8 weeks, the newly synthesized tissue was composed of a fibrous mesh including some cells. However, at 12 weeks it showed a hypercellular hyaline‐like region. This hypercellular region showed excellent bonding with the native cartilage, cells were located in numerous lacunae, and a high content of proteoglycans as indicated by an intense toluidine blue stain was observed. The repaired tissue showed positive immunostaining for both type I and II collagen, as well as chondroitin‐4‐sulfate, chondroitin‐6‐sulfate, keratan sulfate, and integrin subunit β1. Positive staining for porcine anti‐CD46 was localized exclusively in the neo‐synthesized tissue. We conclude that xenotransplantation of pig chondrocytes can repair, in an in vitro model, defects in human articular cartilage.

Human cartilage engineering in an in vitro repair model using collagen scaffolds and mesenchymal stromal cells

The purpose of this study was to investigate cartilage repair of in vitro lesion models using human bone marrow mesenchymal stromal cells (hBMSCs) with different collagen (Col) scaffolds. Lesions were made in human cartilage biopsies. Injured samples were pre-treated with interleukin 1β (IL1β) for 24 h; also, samples were not pre-treated. hBMSCs were seeded on different types of collagen scaffolds. The resulting constructs were placed into the lesions, and the biopsies were cultured for 2 months in chondrogenic medium. Using the modified ICRSII scale, neotissues from the different scaffolds showed ICRS II overall assessment scores ranging from 50% (fibrocartilage) to 100% (hyaline cartilage), except for the Col I +Col II +HS constructs (fibrocartilage/hyaline cartilage, 73%). Data showed that hBMSCs cultured only on Col I +Col II +HS scaffolds displayed a chondrocyte-like morphology and cartilage-like matrix close to native cartilage. Furthermore, IL1β pre-treated biopsies decreased capacity for repair by hBMSCs and decreased levels of chondrogenic phenotype of human cartilage lesions.

Human Amniotic Membrane: A Potential Tissue and Cell Source for Cell Therapy and Regenerative Medicine

The human amniotic membrane (HAM) is the innermost membrane surrounding the fetus. HAM is a highly abundant and readily available tissue that is becoming appreciated as an alternative to adult bone marrow mesenchymal stem cells (BM-MSCs) useful for cell therapy and regenerative medicine. This tissue provides high efficiency in noninvasive and safe MSC recovery with no intrusive procedures. HAM contains two cell types from different embryological origins: human amnion epithelial cells (hAECs), derived from the embryonic ectoderm, and human amnion mesenchymal stromal cells (hAMSCs), derived from the embryonic mesoderm. hAMSCs and hAECs are immune-privileged cells that can be isolated without the sacrifice of human embryos, avoiding immunological rejection problems and the ethical conflict of using human embryonic stem cells (hESCs). Regarding their immunophenotype, both cell types demonstrate the expression of the common well-defined human mesenchymal and embryonic stem cell markers and the absence of hematopoietic markers. Moreover, both cell populations have similar multipotential for in vitro differentiation into all three germ layers: ectoderm, mesoderm, and endoderm lineages. Indeed, the potential application of amnion-derived cells in a variety of diseases, in particular those associated with degenerative processes, is under clinical or preclinical investigation. The HAM has other biological properties important for tissue engineering, including anti-fibrosis, anti-inflammatory, anti-scarring, antimicrobial, as well as adequate mechanical properties and low immunogenicity. Therefore, amnion allografts are widely applied in ophthalmology, plastic surgery, dermatology, and gynecology. In this chapter, the localization, isolation, characterization, and differentiation potential of amnion-derived cells are discussed. Moreover, the potential clinical applications of either amnion-derived cells or the whole HAM are also reviewed.