Simone Elkjær Riis

Critical steps in the isolation and expansion of adipose-derived stem cells for translational therapy

Since the discovery of adipose-derived stem cells (ASCs), there have been high expectations of their putative clinical use. Recent advances support these expectations, and it is expected that the transition from pre-clinical and clinical studies to implementation as a standard treatment modality is imminent. However ASCs must be isolated and expanded according to good manufacturing practice guidelines and a basic assurance of quality, safety, and medical effectiveness is needed for authorisation by regulatory agencies, such as European Medicines Agency and US Food and Drug Administration. In this review, a collection of studies investigating the influence of different steps of the isolation and expansion protocol on the yield and functionality of ASCs has been presented in an attempt to come up with best recommendations that ensure potential beneficial clinical outcome of using ASCs in any therapeutic setting. If the findings confirm the initial observations of beneficial effects of ASCs, the path is paved for implementing these ASC-based therapies as standard treatment options.

Activation of protease-activated receptor 2 induces VEGF independently of HIF-1

Background Human adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible factor 1(HIF-1). The present study hypothesized that PAR2 stimulation through activation of kinase signaling cascades lead to induction of HIF-1 and secretion of VEGF. Methodology/Principal Findings Immunohistochemistry revealed the expression of PAR2 receptors on the surface of hASCs. Blocking the PAR2 receptors with a specific antibody prior to trypsin treatment showed these receptors are involved in trypsin-evoked increase in VEGF secretion from hASCs. Blocking with specific kinase inhibitors suggested that that activation of MEK/ERK and PI3-kinase/Akt pathways are involved in trypsin-eveoked induction of VEGF. The effect of the trypsin treatment on the transcription of VEGF peaked at 6 hours after the treatment and was comparable to the activation observed after keeping hASCs for 24 hours at 1% oxygen. In contrast to hypoxia, trypsin alone failed to induce HIF-1 measured with ELISA, while the combination of trypsin and hypoxia had an additive effect on both VEGF transcription and secretion, results which were confirmed by Western blot. Conclusion In hASCs trypsin and hypoxia induce VEGF expression through separate pathways.

Effect of unaccustomed eccentric exercise on proprioception of the knee in weight and non-weight bearing tasks

The study investigates the effects of eccentric exercise of the quadriceps on proprioception of the knee in weight and non-weight bearing tasks. Proprioception of the exercised leg was assessed at 120° and 150° of knee extension in 15 healthy adults (age 25.0 ± 3.6 yrs) before, immediately after, and 24h following eccentric exercise of the quadriceps. Three tests of proprioception were performed: 1. matching the position of the exercised leg (right leg) to the reference leg (left leg) in sitting (non-weight bearing matching task); 2. repositioning the exercised leg after active movement in sitting (non-weight bearing repositioning task); 3. repositioning the exercised leg after active movement in standing (weight bearing task). Maximum knee extension force was reduced by 77.0 ± 12.3 % immediately after the exercise, and by 82.7 ± 16.2% 24h post exercise, with respect to baseline (P<0.001). The absolute error in the non-weight bearing matching task at 120° of knee extension was greater immediately following eccentric exercise (12.3 ± 5.6, P<0.001) and 24h after exercise (8.1 ± 4.5, P<0.05) compared to baseline (5.8 ± 2.7). Similarly, the absolute error in the non-weight bearing repositioning task at 120° was greater both immediately (5.9 ± 3.1°, P<0.01) and 24h post exercise (5.2 ± 3.0°, P<0.05) compared to baseline (4.5 ± 2.6°). Therefore, in both non-weight bearing tasks, the subjects matched the position of their leg after eccentric exercise by adopting a more extended knee position of the exercised limb. Furthermore, the subjects showed higher variability in their performance immediately post exercise (P<0.05, compared to baseline) but not 24h after. In contrast, eccentric exercise did not affect the repositioning errors in the weight bearing task. In conclusion, eccentric exercise of the quadriceps impairs proprioception of the knee both immediately after and 24h post exercise, but only in non-weight bearing tasks.

Comparative Analysis of Media and Supplements on Initiation and Expansion of Adipose-Derived Stem Cells

Adipose‐derived stem cells (ASCs) are being tested in clinical trials related to cell‐based regenerative therapies. Although most of the current expansion protocols for ASCs use fetal calf serum (FCS), xenogeneic‐free medium supplements are greatly desired. This study aims to compare the effect of FCS, human platelet lysate (hPL), and a fully defined medium on the initiation and maintenance of ASC cultures. ASCs obtained from five donors were cultured in five different media: StemPro, Dulbeccos modified Eagles medium (DMEM) supplemented with 10% hPL, or α‐minimum essential medium (A‐MEM) supplemented with 5% hPL, 10% hPL, or 10% FCS. The effect of media on proliferation, colony‐forming units (CFUs), attachment, and morphology was assessed along with cell size, granularity, and immunophenotype. StemPro greatly compromised the initiation of ASC cultures, which could not survive more than a few passages. Cells cultured in A‐MEM proliferated at a faster rate than in DMEM, and hPL significantly enhanced cell size, granularity, and proliferation compared with FCS. All media except StemPro supported CFUs equally well. Analysis of surface markers revealed higher levels of CD73 and CD105 in FCS‐cultured ASCs, whereas increased levels of CD146 were found in hPL‐cultured cells. Multiparametric flow cytometric analysis performed after seven passages revealed the existence of four distinct ASC subpopulations, all positive for CD73, CD90, and CD105, which mainly differed by their expression of CD146 and CD271. Analysis of the different subpopulations might represent an important biological measure when assessing different medium formulations for a particular clinical application.

Implications of Extracellular Matrix Production by Adipose Tissue-Derived Stem Cells for Development of Wound Healing Therapies

The synthesis and deposition of extracellular matrix (ECM) plays an important role in the healing of acute and chronic wounds. Consequently, the use of ECM as treatment for chronic wounds has been of special interest—both in terms of inducing ECM production by resident cells and applying ex vivo produced ECM. For these purposes, using adipose tissue-derived stem cells (ASCs) could be of use. ASCs are recognized to promote wound healing of otherwise chronic wounds, possibly through the reduction of inflammation, induction of angiogenesis, and promotion of fibroblast and keratinocyte growth. However, little is known regarding the importance of ASC-produced ECM for wound healing. In this review, we describe the importance of ECM for wound healing, and how ECM production by ASCs may be exploited in developing new therapies for the treatment of chronic wounds.

Hypoxia enhances the wound-healing potential of adipose-derived stem cells in a novel human primary keratinocyte-based scratch assay

Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low calcium content compromised the growth of ASCs. We found that it is possible to perform the wound healing assay with primary keratinocytes, if the conditioned media from the ASCs is dialyzed to reduce the calcium concentration. Additionally, using this model of re-epithelization, conditioned media from normoxic ASCs was shown to markedly increase the rate of wound closure by primary keratinocytes, and this effect was significantly enhanced with media from the hypoxia-exposed ASCs. These findings, which are in line with the observations from previous in vivo studies, highlight the validity of this modified assay to investigate the wound healing properties of ASCs in vitro.

Discrete adipose-derived stem cell subpopulations may display differential functionality after in vitro expansion despite convergence to a common phenotype distribution.

BackgroundComplex immunophenotypic repertoires defining discrete adipose-derived stem cell (ASC) subpopulations may hold a key toward identifying predictors of clinical utility. To this end, we sorted out of the freshly established ASCs four subpopulations (SPs) according to a specific pattern of co-expression of six surface markers, the CD34, CD73, CD90, CD105, CD146, and CD271, using polychromatic flow cytometry.MethodUsing flow cytometry-associated cell sorting and analysis, gating parameters were set to select for a CD73+CD90+CD105+ phenotype plus one of the four following combinations, CD34−CD146−CD271− (SP1), CD34−CD146+CD271− (SP2), CD34+CD146+CD271− (SP3), and CD34−CD146+CD271+ (SP4). The SPs were expanded 700- to 1000-fold, and their surface repertoire, trilineage differentiation, and clonogenic potential, and the capacity to support wound healing were assayed.ResultsUpon culturing, the co-expression of major epitopes, the CD73, CD90, and CD105 was maintained, while regarding the minor markers, all SPs reverted to resemble the pre-sorted population with CD34−CD146−CD271− and CD34−CD146+CD271− representing the most prevalent combinations, followed by less frequent CD34+CD146−CD271− and CD34+CD146+CD271− variants. There was no difference in the efficiency of adipo-, osteo-, or chondrogenesis by cytochemistry and real-time RT-PCR or the CFU capacity between the individual SPs, however, the SP2CD73+90+105+34-146+271- outperformed others in terms of wound healing.ConclusionsOur study shows that ASCs upon culturing inherently maintain a stable distribution of immunophenotype variants, which may potentially disguise specific functional properties of particular downstream lines. Furthermore, the outlined approach suggests a paradigm whereby discrete subpopulations could be identified to provide for a therapeutically most relevant cell product.

Pigmentation is associated with stemness hierarchy of progenitor cells within cultured limbal epithelial cells

Ex vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness‐associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence‐activated cell sorting and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co‐expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3. A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co‐expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. Stem Cells 2018;36:1411–1420