Interfollicular Epidermal Stem Cells: Boosting and Rescuing from Adult Skin.

Epidermal stem cells (EpSCs) isolation struggle remains, mainly due to the yet essential requirement of well-defined approaches and markers. The herein proposed methodology integrates an assemblage of strategies to accomplish the enrichment of the interfollicular EpSCs multipotent fraction and their subsequent separation from the remaining primary human keratinocytes (hKC) culture. Those include rapid adherence of freshly isolated hKC to collagen type IV through the ß1-integrin ligand and Rho-associated protein kinase inhibitor (Rocki) Y-27632 administration to the cultures, followed by an immunomagnetic separation to obtain populations based in the combined CD49fbri/CD71dim expression. Flow cytometry is the supporting method to analyze the effect of the treatments over the expression rate of early epidermal markers keratins19/5/14 and in correlation to CD49fbri/CD71dim sub-populations. The step-by-step methodology herein described indulges the boosting and consecutive purification and separation of interfollicular epidermal stem cells, from human keratinocytes cultures.

In vitro and in vivo performance of methacrylated gellan gum hydrogel formulations for cartilage repair.

Methacrylated gellan gum (GGMA) formulation is proposed as a second-generation hydrogel for controlled delivery of cartilage-forming cells into focal chondral lesions, allowing immediate in situ retention of cells and 3D filling of lesion volume, such approach deemed compatible with an arthroscopic procedure. Formulation optimization was carried out in vitro using chondrocytes and adipose mesenchymal stromal/stem cells (ASCs). A proof-of-concept in vivo study was conducted using a rabbit model with induced chondral lesions. Outcomes were compared with microfracture or non-treated control. Three grading scores were used to evaluate tissue repair after 8 weeks by macroscopic, histological and immunohistochemical analysis. Intense collagen type II and low collagen type I gene and protein expression were achieved in vitro by the ASC + GGMA formulation, in light with development of healthy chondral tissue. In vivo, this formulation promoted significantly superior de novo cartilage formation compared with the non-treated group. Maintenance of chondral height and integration with native tissue was further accomplished. The physicochemical properties of the proposed GGMA hydrogel exhibited highly favorable characteristics and biological performance both in vitro and in vivo, positioning itself as an attractive xeno-free biomaterial to be used with chondrogenic cells for a cost-effective treatment of focal chondral lesions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1987-1996, 2018.

Boosting and rescuing epidermal superior population from fresh keratinocyte cultures.

Epidermal stem cells (EpSCs) hold great expectations in a regenerative medicine context, but innovative methods that permit to obtain a significant yield of EpSCs or stem-like epidermal cells are still required. We propose a two-step strategy to obtain a superior epidermal stem-like cell fraction among primary keratinocytes (KCs) isolated from adult human skin. The approach is based on the combination of rapid adherence to collagen IV with the rock-associated kinase inhibitor (ROCKi) treatment, and the subsequent immunomagnetic separation of the α6(high)/CD71(dim) cell subset. The combined collagen IV and ROCKi treatment showed not only to enhance cells clonogenic capacity, but also to induce an early epidermal phenotypic signature, along with the diminished expression of late differentiation-associated markers. More importantly, collagen IV and the ROCKi efficiently promoted a synergized effect over α6(high)/CD71(dim) expression, boosting the number of highly proliferative KCs stem-like cells as demonstrated by the expression of ki67. This cell fraction showed a superior ability to generate a 3D stratified epithelium formed by cells with successive differentiation phenotypes. Overall, this strategy indulged the possibility to uncover, among adult KCs, a superior epidermal cell population with stem-like proliferation capacity and early differentiation degree to be used in numerous skin regeneration approaches.

Interfollicular epidermal stem cells: boosting and rescuing from adult skin.

Epidermal stem cells isolation struggle remains, mainly due to the yet essential requirement of well-defined approaches and markers. The herein proposed methodology integrates an assemblage of strategies to accomplish the enrichment of the interfollicular epidermal stem cells multipotent fraction and their subsequent separation from the remaining primary human keratinocytes culture. Those include rapid adherence of freshly isolated human keratinocytes to collagen type IV through the ß1-integrin ligand and Rho-Associated Protein Kinase Inhibitor Y- 27632 administration to the cultures, followed by an immunomagnetic separation to obtain populations based in the combined CD49f(bri)/CD71(dim) expression. Flow cytometry is the supporting method to analyze the effect of the treatments over the expression rate of early epidermal markers keratins19/5/14 and in correlation to CD49f(bri)/CD71(dim) subpopulations. The step-by-step methodology herein described indulges the boosting and consecutive purification and separation of interfollicular epidermal stem cells from human keratinocytes cultures.

Human adipose tissue-derived SSEA-4 subpopulation multi-differentiation potential towards the endothelial and osteogenic lineages.

Human adipose tissue has been recently recognized as a potential source of stem cells for regenerative medicine applications, including bone tissue engineering (TE). Despite the gathered knowledge regarding the differentiation potential of human adipose tissue-derived stem cells (hASCs), in what concerns the endothelial lineage many uncertainties are still present. The existence of a cell subpopulation within the human adipose tissue that expresses a SSEA-4 marker, usually associated to pluripotency, raises expectations on the differentiation capacity of these cells (SSEA-4(+)hASCs). In the present study, the endothelial and osteogenic differentiation potential of the SSEA-4(+)hASCs was analyzed, aiming at proposing a single-cell source/subpopulation for the development of vascularized bone TE constructs. SSEA-4(+)hASCs were isolated using immunomagnetic sorting and cultured either in α-MEM, in EGM-2 MV (endothelial growth medium), or in osteogenic medium. SSEA-4(+)hASCs cultured in EGM-2 MV formed endothelial cell-like colonies characterized by a cobblestone morphology and expression of CD31, CD34, CD105, and von Willebrand factor as determined by quantitative reverse transcriptase (RT)-polymerase chain reaction, immunofluorescence, and flow cytometry. The endothelial phenotype was also confirmed by their ability to incorporate acetylated low-density lipoprotein and to form capillary-like structures when seeded on Matrigel. SSEA-4(+)hASCs cultured in α-MEM displayed fibroblastic-like morphology and exhibited a mesenchymal surface marker profile (>90% CD90(+)/CD73(+)/CD105(+)). After culture in osteogenic conditions, an overexpression of osteogenic-related markers (osteopontin and osteocalcin) was observed both at molecular and protein levels. Matrix mineralization detected by Alizarin Red staining confirmed SSEA-4(+)hASCs osteogenic differentiation. Herein, we demonstrate that from a single-cell source, human adipose tissue, and by selecting the appropriate subpopulation it is possible to obtain microvascular-like endothelial cells and osteoblasts, the most relevant cell types for the creation of vascularized bone tissue-engineered constructs.

Perivascular-like cells contribute to the stability of the vascular network of osteogenic tissue formed from cell sheet-based constructs.

In recent years several studies have been supporting the existence of a close relationship in terms of function and progeny between Mesenchymal Stem Cells (MSCs) and Pericytes. This concept has opened new perspectives for the application of MSCs in Tissue Engineering (TE), with special interest for the pre-vascularization of cell dense constructs. In this work, cell sheet technology was used to create a scaffold-free construct composed of osteogenic, endothelial and perivascular-like (CD146(+)) cells for improved in vivo vessel formation, maturation and stability. The CD146 pericyte-associated phenotype was induced from human bone marrow mesenchymal stem cells (hBMSCs) by the supplementation of standard culture medium with TGF-ß1. Co-cultured cell sheets were obtained by culturing perivascular-like (CD146(+)) cells and human umbilical vein endothelial cells (HUVECs) on an hBMSCs monolayer maintained in osteogenic medium for 7 days. The perivascular-like (CD146(+)) cells and the HUVECs migrated and organized over the collagen-rich osteogenic cell sheet, suggesting the existence of cross-talk involving the co-cultured cell types. Furthermore the presence of that particular ECM produced by the osteoblastic cells was shown to be the key regulator for the singular observed organization. The osteogenic and angiogenic character of the proposed constructs was assessed in vivo. Immunohistochemistry analysis of the explants revealed the integration of HUVECs with the host vasculature as well as the osteogenic potential of the created construct, by the expression of osteocalcin. Additionally, the analysis of the diameter of human CD146 positive blood vessels showed a higher mean vessel diameter for the co-cultured cell sheet condition, reinforcing the advantage of the proposed model regarding blood vessels maturation and stability and for the in vitro pre-vascularization of TE constructs.

Dynamic culturing of cartilage tissue: the significance of hydrostatic pressure.

Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and amplitude-dependant manner.

Tissue engineering and regenerative medicine strategies in meniscus lesions.

PURPOSE: The aim of this systematic review was to address tissue engineering and regenerative medicine (TERM) strategies applied to the meniscus, specifically (1) clinical applications, indications, results, and pitfalls and (2) the main trends in research assessed by evaluation of preclinical (in vivo) studies. METHODS: Three independent reviewers performed a search on PubMed, from 2006 to March 31, 2011, using the term "meniscus" with all of the following terms: "scaffolds," "constructs," "cells," "growth factors," "implant," "tissue engineering," and "regenerative medicine." Inclusion criteria were English language-written, original clinical research (Level of Evidence I to IV) and preclinical studies of TERM application in knee meniscal lesions. Reference lists and related articles on journal Web sites of selected articles were checked until prepublication for potential studies that could not be identified eventually by our original search. The modified Coleman Methodology score was used for study quality analysis of clinical trials. RESULTS: The PubMed search identified 286 articles (a similar search from 2000 to 2005 identified 161 articles). Non-English-language articles (n = 9), Level V publications (n = 19), in vitro studies (n = 118), and 102 studies not related to the topic were excluded. One reference was identified outside of PubMed. Thirty-eight references that met the inclusion criteria were identified from the original search. On the basis of our prepublication search, 2 other references were included. A total of 9 clinical and 31 preclinical studies were selected for further analysis. Of the clinical trials, 1 was classified as Level I, 2 as Level II, and 6 as Level IV. Eight referred to acellular scaffold implantation for partial meniscal replacement, and one comprised fibrin clot application. The mean modified Coleman Methodology score was 48.0 (SD, 15.7). Of the preclinical studies, 11 original works reported on studies using large animal models whereas 20 research studies used small animals. In these studies the experimental design favored cell-seeded scaffolds or scaffolds enhanced with growth factors (GFs) in attempts to improve tissue healing, as opposed to the plain acellular scaffolds that were predominant in clinical trials. Injection of mesenchymal stem cells and gene therapy are also presented as alternative strategies. CONCLUSIONS: Partial meniscal substitution using acellular scaffolds in selected patients with irreparable loss of tissue may be a safe and promising procedure. However, there is only 1 randomized controlled study supporting its application, and globally, many methodologic issues of published trials limit further conclusions. We registered a different trend in preclinical trials, with most considering augmentation of scaffolds by cells and/or GFs, as opposed to the predominantly acellular approach in clinical trials. Different TERM approaches to enhance meniscal repair or regeneration are in preclinical analysis, such as the use of mesenchymal stem cells, gene therapy, and GFs alone or in combination, and thus could be considered in the design of subsequent trials. LEVEL OF EVIDENCE: Level IV, systematic review of Level I to IV studies.

Synthesis and functionalization of superparamagnetic poly-ε-caprolactone microparticles for the selective isolation of subpopulations of human adipose-derived stem cells.

There has been a growing interest in using biofunctionalized magnetic particles for cell isolation. This paper describes the synthesis and characterization of magnetite-polymer (Fe(3)O(4)-poly-ε-caprolactone, magnetite-PCL) microparticles surface functionalized with amino and epoxy groups allowing easy covalent attachment of specific antibodies and subsequent ability to bind target cells. Particles with different sizes (4-135 µm), spherical shape and superparamagnetic behaviour (magnetite content of about 13 wt%) were obtained. The functionalized microparticles presented high protein-binding capacity (coupling efficiency of 47% for epoxy- and 71% for amino-functionalized particles) with a low level of non-specific binding. We have further investigated the influence of initial protein concentration, pH, ionic strength, temperature and incubation time on the capacity of amino-functionalized particles to bind protein molecules. The results showed that maximum protein coupling is rapidly achieved (≤5 h) at pH 5.5 and low ionic strength (0.05 M NaCl). Furthermore, when cultured in direct contact with osteoblast-like cells (Saos-2) or human-derived adipose stem cells (ASCs), the amino-functionalized particles did not affect the proliferation and morphology of the cells. As a proof of principle for the application of magnetic microparticles for cell isolation, CD105 (endoglin) antibody was coupled to the magnetic particle surface to bind subpopulations of human ASCs expressing the CD105 antigen. The isolation of CD105+ ASCs from a heterogeneous cell population was confirmed by flow cytometry analysis. Given the demonstrated potential of functionalized magnetite-PCL microparticles for selective cell isolation, we expect that these particles may be further applied in immuno-magnetic cell separation owing to their versatility and ease of surface modification.

Carrageenan-based hydrogels for the controlled delivery of PDGF-BB in bone tissue engineering applications.

One of the major drawbacks found in most bone tissue engineering approaches developed so far consists in the lack of strategies to promote vascularisation. Some studies have addressed different issues that may enhance vascularisation in tissue engineered constructs, most of them involving the use of growth factors (GFs) that are involved in the restitution of the vascularity in a damaged zone. The use of sustained delivery systems might also play an important role in the re-establishment of angiogenesis. In this study, kappa-carrageenan, a naturally occurring polymer, was used to develop hydrogel beads with the ability to incorporate GFs with the purpose of establishing an effective angiogenesis mechanism. Some processing parameters were studied and their influence on the final bead properties was evaluated. Platelet derived growth factor (PDGF-BB) was selected as the angiogenic factor to incorporate in the developed beads, and the results demonstrate the achievement of an efficient encapsulation and controlled release profile matching those usually required for the development of a fully functional vascular network. In general, the obtained results demonstrate the potential of these systems for bone tissue engineering applications.

Generation of functional natural killer and dendritic cells in a human stromal-based serum-free culture system designed for cord blood expansion.

OBJECTIVE: We have previously reported on the ability of a mesenchymal stem cell-based serum-free culture system to expand human cord blood (CB) hematopoietic stem cells along the myeloid pathway and simultaneously generate a CD7(+)CD34(-) population. In this study, we investigated the ability of the CD7(+)CD34(-) population to differentiate into natural killer and dendritic cells (DCs). MATERIALS AND METHODS: CB CD34(+) cells were expanded over a mesenchymal stem cell layer in serum-free medium supplemented with stem cell factor, basic fibroblast growth factor, leukemia inhibitor factor, and Flt-3 ligand for 2 weeks. Cultured cells were harvested and CD7(+)CD34(-)Lin(-) cells sorted and plated for 2 additional weeks in either natural killer- or DC-inductive medium. RESULTS: Culture of CD34(+) cells for the first 2 weeks in this system resulted in expansion of the stem cell pool and the myeloid component of the graft, and also produced a 58-fold increase in the CD7(+)CD34(-) cell population. When sorted CD7(+)CD34(-)Lin(-) cells were induced toward a natural killer cell phenotype, further expansion was observed during this time in culture, and differentiation was confirmed by cytotoxic activity and by flow cytometry, with cells displaying CD16 and CD56 in the absence of CD3. Generation of DC cells in culture was also verified by observing both the characteristic dendritic morphology and the dendritic phenotypes HLA-DR(bright)CD123(bright)CD11c(-) and HLA-DR(bright)CD11c(+). CONCLUSION: These results demonstrate the ability of an ex vivo culture system to drive expansion of human CB hematopoietic stem cells, while promoting the immune maturation of the graft and generation of DC and natural killer cells that could then be utilized for adoptive cancer cellular immunotherapy.