Characterization of Adult Canine Kidney Epithelial Stem Cells That Give Rise to Dome-Forming Tubular Cells.

Dome formation can occur in cultured tubular epithelial cells originating from various tissues, including the mammary gland and the kidney. The isolation and characterization of normal kidney epithelial stem cells that give rise to dome-forming tubular cells have never been reported. We attempted to isolate and characterize canine kidney epithelial stem cells using a simple cell culture method that we have previously used to isolate other adult human stem cells. Dome-forming kidney epithelial cells were derived from dissociated adult canine kidney tissues that were cultured in a modified keratinocyte serum-free medium supplemented with N-acetyl-l-cysteine, l-ascorbic acid 2-phosphate, nicotinamide, and fetal bovine serum. These cells exhibited high self-renewal capacity in long-term culture (growth for >13 months and 30 cumulative population doublings) and exhibited characteristics of stem cells, including (1) deficiency in gap junctional intercellular communication, (2) anchorage-independent growth, (3) expression of stem cell markers octamer-binding transcription factor 4 and SRY (sex determining region Y)-box 2, (4) expression of cell surface markers CD24 and CD133, and (5) multipotent differentiation into osteoblasts, adipocytes, chondrocytes, and dome-forming tubular cells. Most of these characteristics are shared by the well-known canine renal tubule-derived immortalized Madin-Darby Canine Kidney cell line. Furthermore, the putative canine kidney stem cells developed in this study formed budding tubule-like organoids on Matrigel and required high cell density (>4,000 cells/cm2) for sustained growth and confluency for dome formation. The signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitor, AG490, inhibited colony-forming efficiency and dome formation, whereas lipopolysaccharide, an activator of STAT3, increased colony-forming efficiency in a dose-dependent manner. These results are consistent with the hypothesis that high cell density induces STAT3 expression, which promotes both stem cell self-renewal and differentiation into tubular cells. Our novel cell culture method should be useful for the future development of normal human kidney stem cells for clinical applications and for studying mechanisms of nephrotoxicity.

MagA expression attenuates iron export activity in undifferentiated multipotent P19 cells.

Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport protein from magnetotactic bacteria, is a useful gene-based magnetic resonance contrast agent. Hemagglutinin-tagged MagA was stably expressed in undifferentiated embryonic mouse teratocarcinoma, multipotent P19 cells to provide a suitable model for tracking these cells during differentiation. Western blot and immunocytochemistry confirmed the expression and membrane localization of MagA in P19 cells. Surprisingly, elemental iron analysis using inductively-coupled plasma mass spectrometry revealed significant iron uptake in both parental and MagA-expressing P19 cells, cultured in the presence of iron-supplemented medium. Withdrawal of this extracellular iron supplement revealed unexpected iron export activity in P19 cells, which MagA expression attenuated. The influence of iron supplementation on parental and MagA-expressing cells was not reflected by longitudinal relaxation rates. Measurement of transverse relaxation rates (R2* and R2) reflected changes in total cellular iron content but did not clearly distinguish MagA-expressing cells from the parental cell type, despite significant differences in the uptake and retention of total cellular iron. Unlike other cell types, the reversible component R2' (R2* ‒ R2) provided only a moderately strong correlation to amount of cellular iron, normalized to amount of protein. This is the first report to characterize MagA expression in a previously unrecognized iron exporting cell type. The interplay between contrast gene expression and systemic iron metabolism substantiates the potential for diverting cellular iron toward the formation of a novel iron compartment, however rudimentary when using a single magnetotactic bacterial gene expression system like magA. Since relatively few mammalian cells export iron, the P19 cell line provides a tractable model of ferroportin activity, suitable for magnetic resonance analysis of key iron-handling activities and their influence on gene-based MRI contrast.

The Haematococcus pluvialis extract enriched by bioaccumulation process with Mg(II) ions improves insulin resistance in equine adipose-derived stromal cells (EqASCs).

Insulin resistance (IR) is one of the characteristic features of equine metabolic syndrome (EMS). Presently, the only therapies of choice are caloric restrictions combined with mineral supplementation, which might improve insulin sensitivity. In this study we investigated the effect of Haematococcus pluvialis algae water extract enriched in bioaccumulation process in magnesium ions (Hp_Mg(II)) on equine adipose derived mesenchymal stromal stem cells, in which insulin resistance was induced by palmitic acid (IR-EqASCs). For this purpose, chemical characterization of H. pluvialis was performed with special emphasis on the analysis of minerals composition, total phenolic and carotenoids contents, as well as scavenging activity. To examine the influence of H. pluvialis extract on IR-EqASCs, various methods of molecular biology and microscopic observations (i.e., immunofluorescence staining, SEM, gene expression by RT-qPCR, proliferative and metabolic cells activity analysis) were applied to investigate in vitro viability, oxidative stress markers and apoptosis-related factor accumulation, along with insulin resistance-related genes expression. Obtained results show, that Hp_Mg(II) significantly improves proliferative and metabolic activity of IR-EqASCs, shortens their population doubling time, improves their clonogenic potential and reduces expression of apoptosis related genes. Moreover, anti-oxidative effect of extract was presented.

Osteogenic differentiation of skeletal muscle progenitor cells is activated by the DNA damage response.

Heterotopic ossification (HO) is a pathological condition characterized by the deposition of mineralized tissue in ectopic locations such as the skeletal muscle. The precise cellular origin and molecular mechanisms underlying HO are still debated. In our study we focus on the differentiation of mesoangioblasts (MABs), a population of multipotent skeletal muscle precursors. High-content screening for small molecules that perturb MAB differentiation decisions identified Idoxuridine (IdU), an antiviral and radiotherapy adjuvant, as a molecule that promotes MAB osteogenic differentiation while inhibiting myogenesis. IdU-dependent osteogenesis does not rely on the canonical BMP-2/SMADs osteogenic pathway. At pro-osteogenic conditions IdU induces a mild DNA Damage Response (DDR) that activates ATM and p38 eventually promoting the phosphorylation of the osteogenesis master regulator RUNX2. By interfering with this pathway IdU-induced osteogenesis is severely impaired. Overall, our study suggests that induction of the DDR promotes osteogenesis in muscle resident MABs thereby offering a new mechanism that may be involved in the ectopic deposition of mineralized tissue in the muscle.

Definition of a cell surface signature for human cardiac progenitor cells after comprehensive comparative transcriptomic and proteomic characterization.

Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC. iTRAQ analysis confirmed differential expression of a substantial proportion of those proteins in CPC relative to MSC, and systems biology analysis defined a clear overrepresentation of several categories related to enhanced angiogenic potential. The CPC plasma membrane compartment comprised 1,595 proteins, including a minimal signature of 167 proteins preferentially or exclusively expressed by CPC. CDH5 (VE-cadherin),  OX2G (OX-2 membrane glycoprotein; CD200), GPR4 (G protein-coupled receptor 4), CACNG7 (calcium voltage-gated channel auxiliary subunit gamma 7) and F11R (F11 receptor; junctional adhesion molecule A; JAM-A; CD321) were selected for validation. Their differential expression was confirmed both in expanded CPC batches and in early stages of isolation, particularly when compared against cardiac fibroblasts. Among them, GPR4 demonstrated the highest discrimination capacity between all cell lineages analyzed.

Additive effect of bFGF and selenium on expansion and paracrine action of human amniotic fluid-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cell-derived conditioned medium (MSC-CM) has emerged as a promising cell-free tool for restoring degenerative diseases and treating traumatic injuries. The present study describes the effect of selenium as a reactive oxygen species (ROS) scavenger and its additive effect with basic fibroblast growth factor (bFGF) on in vitro expansion of amniotic fluid (AF)-MSCs and the paracrine actions of AF-MSC-CM as well as the associated cellular and molecular mechanisms. METHODS: In this study, we obtained CM from human AF-MSCs cultured with selenium. The stemness of selenium-treated AF-MSCs was evaluated by cell growth and differentiation potential. Human fibroblasts were treated with AF-MSC-CM and analyzed for cell signaling changes. For in vivo wound healing assay, ICR mice with a full-thickness skin wound were used. RESULTS: Selenium played a critical role in in vitro expansion of AF-MSCs through activation of the AKT-ERK1/2, Smad2, and Stat3 signaling pathways along with inactivation of GSK3ß. When administered together with bFGF, it showed remarkable effect in inhibiting ROS accumulation and preserving their multipotency. Proliferation and migration of human dermal fibroblasts and in vivo wound healing were improved in the CMs derived from AF-MSCs exposed to selenium and bFGF, which was caused by the Smad2, AKT-MEK1/2-ERK, and NFκB signaling triggered by the paracrine factors of AF-MSCs, such as TGF-ß, VEGF, and IL-6. Our results suggest the following: (a) supplementation of selenium in AF-MSC culture contributes to in vitro expansion and preservation of multipotency, (b) ROS accumulation causes progressive losses in proliferative and differentiation potential, (c) the separate activities of bFGF and selenium in MSCs exert an additive effect when used together, and (d) the additive combination improves the therapeutic effects of AF-MSC-derived CMs on tissue repair and regeneration. CONCLUSION: Antioxidants, such as selenium, should be considered as an essential supplement for eliciting the paracrine effects of MSC-CMs.

Three-Dimensional Ameliorated Biologics Elicit Thymic Renewal in Tumor-Bearing Hosts.

Cancer-initiating/sustaining stem cell subsets (CSCs) have the potential to regenerate cancer cell populations and are resistant to routine therapeutic strategies, thus attracting much attention in anticancer research. In this study, an innovative framework of endogenous microenvironment-renewal for addressing such a dilemma has been just developed. CSCs in three-dimensional multipotent spheroid-engineered biologics were prepared with 150 Gy radiation and inoculated into 15-mo-old BALB/c and C57BL/6 mice bearing diverse advanced tumors covering Mammary 4T1, liver Hepa, lung LL/2, and colon C26 tumors and distant metastases. Subsequently, the systematic microenvironment of tumor-bearing hosts was rapidly remodeled to resettle thymic cortex and medulla rudiment as an endogenous foxn1-thymosin reprogramming TCR-repertoire for resetting MHC-unrestricted multifunction renewal. Postrenewal Vγ4γδT-subsets would bind and lead migrating CSCs into apoptosis. Moreover, TCR repertoire multifunction renewal could reverse tumor metastases from tumoricidal resistance into eventual regression as a blockade of cancer-sustaining Bmi-1/Nanog-Oct4-Sox2 renewal loop with sequent multivalent depletion of both migrating/in situ CSCs and non-stem terminal cancer cell subsets. This study represents a promising start to set up a generalizable strategy of three-dimensional biologics evoking an endogenous integral microenvironment into pluripotent renewal versus advanced cancer.

The assessment of multipotent cell transplantation in acute-on-chronic liver failure: a systematic review and meta-analysis.

Acute-on-chronic liver failure (ACLF) is a serious life-threatening disease with high prevalence. Liver transplantation is the only efficient clinical treatment for ACLF. Because of the rapid progression and lack of liver donors, it is urgent to find an effective and safe therapeutic approach to ACLF. Recent studies showed that multipotent cell transplantation could improve the patients' liver function and enhance their preoperative condition. Cells such as mesenchymal stem cells, bone marrow mononuclear cells and autologous peripheral blood stem cells, which addressed in this study have all been used in multipotent cell transplantation for liver diseases. However, its clinical efficiency is still debatable. This systematic review and meta-analysis explored the clinical efficiency of multipotent cell transplantation as a therapeutic approach for patients with ACLF. A detailed search of the Cochrane Library, MEDLINE, and Embase databases was conducted from inception to November 2017. The outcome measures were serum albumin, prothrombin time, alanine aminotransferase, total bilirubin, platelets, hemoglobin, white blood cells, and survival time. The quality of evidence was assessed using GRADEpro and Jaded scores. A literature search resulted in 537 citations. Of these, 9 articles met the inclusion criteria. It was found that multipotent cell transplantation was able to alleviate liver damage and improve liver function. Multipotent cell transplantation can also enhance the short-term and medium-term survival rates of ACLF. All 9 research articles included in this analysis reported no statistically significant adverse events, side effects, or complications. In conclusions, this study suggested that multipotent cell transplantation could be recommended as a potential therapeutic supplementary tool in clinical practice. However, clinical trials in large-volume centers still needed.

Palovarotene inhibits connective tissue progenitor cell proliferation in a rat model of combat-related heterotopic ossification.

Heterotopic ossification (HO) develops in the extremities of wounded service members and is common in the setting of high-energy penetrating injuries and blast-related amputations. No safe and effective prophylaxis modality has been identified for this patient population. Palovarotene has been shown to reduce bone formation in traumatic and genetic models of HO. The purpose of this study was to determine the effects of Palovarotene on inflammation, progenitor cell proliferation, and gene expression following a blast-related amputation in a rodent model (n = 72 animals), as well as the ability of Raman spectroscopy to detect early HO before radiographic changes are present. Treatment with Palovarotene was found to dampen the systemic inflammatory response including the cytokines IL-6 (p = 0.01), TNF-α (p = 0.001), and IFN-γ (p = 0.03) as well as the local inflammatory response via a 76% reduction in the cellular infiltration at post-operative day (POD)-7 (p = 0.03). Palovarotene decreased osteogenic connective tissue progenitor (CTP-O) colonies by as much as 98% both in vitro (p = 0.04) and in vivo (p = 0.01). Palovarotene treated animals exhibited significantly decreased expression of osteo- and chondrogenic genes by POD-7, including BMP4 (p = 0.02). Finally, Raman spectroscopy was able to detect differences between the two groups by POD-1 (p < 0.001). These results indicate that Palovarotene inhibits traumatic HO formation through multiple inter-related mechanisms including anti-inflammatory, anti-proliferative, and gene expression modulation. Further, that Raman spectroscopy is able to detect markers of early HO formation before it becomes radiographically evident, which could facilitate earlier diagnosis and treatment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1135-1144, 2018.

Three dimensional de novo micro bone marrow and its versatile application in drug screening and regenerative medicine.

The finding that bone marrow hosts several types of multipotent stem cell has prompted extensive research aimed at regenerating organs and building models to elucidate the mechanisms of diseases. Conventional research depends on the use of two-dimensional (2D) bone marrow systems, which imposes several obstacles. The development of 3D bone marrow systems with appropriate molecules and materials however, is now showing promising results. In this review, we discuss the advantages of 3D bone marrow systems over 2D systems and then point out various factors that can enhance the 3D systems. The intensive research on 3D bone marrow systems has revealed multiple important clinical applications including disease modeling, drug screening, regenerative medicine, etc. We also discuss some possible future directions in the 3D bone marrow research field.

Investigation of growth conditions for the expansion of porcine mesenchymal stem cells on microcarriers in stirred cultures.

The extensive use of mesenchymal stem cells (MCS) in tissue engineering and cell therapy increases the necessity to improve their expansion. Among these, porcine MCS are valuable models for tissue engineering and are classically expanded in static T-flasks. In this work, different processes of stirred cultures were evaluated and compared. First, the effect of glucose, glutamine, antioxidant, and growth factors concentrations on porcine MSC expansion were analyzed in a suitable medium by performing kinetic studies. Results showed that a lower glucose concentration (5.5 mM) enabled to increase maximal cell concentration by 40 % compared with a higher one (25 mM), while addition of 2 to 6 mM of glutamine increased maximal cell concentration by more than 25 % compared with no glutamine supplementation. Moreover, supplementation with 1 µM thioctic acid increased maximal cell concentration by 40 % compared with no supplementation. Using this adapted medium, microcarriers cultures were performed and compared with T-flasks expansion. Porcine MSC were shown to be able to proliferate on the five types of microcarriers tested. Moreover, cultures on Cytodex 1, Cytopore 2, and Cultispher G exhibited a MSC growth rate more than 40 % higher compared with expansion in T-flasks, while MSC metabolism was similar.

Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats.

The design of bioactive three-dimensional (3D) scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2) influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs) was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs) and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.

Influence of pressurized cyclic stretch and endothelial cell presence on multipotent stem cell osteogenic commitment.

Applied mechanical stretch and blood vessel invasion are key stimuli to which progenitor cells are exposed in post-natal endochondral bone formation. Understanding the combined effects of cyclic stretch and endothelial cell (EC) presence on multipotent stem cell (MSC) osteogenesis therefore has the potential to lead to improved MSC-based bone regeneration strategies. Toward this goal, 10T1/2 mouse MSCs were encapsulated in tubular poly(ethylene glycol) diacrylate [PEGDA] hydrogels with moduli within the "osteogenic" range in order to induce osteogenesis. Half of the constructs were fabricated with a luminal EC layer. All of the EC(+) (EC(+)/dyn(+)) and half of the EC(-) constructs (EC(-)/dyn(+)) were subjected to pressurized cyclic stretch in the absence of osteogenic media supplements, with remaining EC(-) constructs (EC(-)/dyn(-)) serving as static controls. At day 10 of culture, expression of the bone extracellular matrix protein osteopontin was over 3.3- and 1.9-fold higher in the EC(+)/dyn(+) and EC(-)/dyn(+) constructs, respectively, relative to day 0. At day 22 of culture, osteopontin levels could not be statistically distinguished from day 0 in the EC(+)/dyn(+) constructs and were one-third less than day 0 in the EC(-)/dyn(+) constructs. In contrast, at day 22 levels of an osteogenic marker alkaline phosphatase (AP) were over 2.4- and 1.4-fold higher in the EC(+)/dyn(+) and EC(-)/dyn(+) constructs, respectively, relative to day 0. Furthermore, at day 22 matrix mineralization in both dynamic groups was increased over 2.5-fold and over 9-fold relative to the EC(-)/dyn(-) and day 0 groups, respectively. Cumulatively, these results suggest that pressurized cyclic stretch alone significantly increases the rate/degree of osteogenesis relative to static culture. However, EC presence combined with pressured cyclic stretch appears to further enhance the rate/degree of MSC osteogenesis and/or to support a distinct osteogenic "fingerprint" compared to that promoted by cyclic stretch alone.

Compound selection for in vitro modeling of developmental neurotoxicity.

Development of in vitro systems, such as those based on embryonic stem cell differentiation, depends on the selection of adequate test and training compounds. We recommend the use of two classes of positive controls, the "gold standard compounds" for which developmental neurotoxicity (DNT) has been proven in man, and the "pathway compounds" that are known to disrupt signalling pathways and key processes relevant for neuronal differentiation. We introduce the concept of toxicity endophenotypes (TEP) as changes in neuronal connectivity resulting from exposure to developmental toxicants. Thus, TEPs provide the scientific rationale for modeling DNT with simple in vitro models of key neurodevelopmental events. In this context, we discuss scientific and technical aspects of the test compound selection process. We suggest to include compounds with unspecific toxicity, besides negative control compounds, and we recommend tandem approaches to determine relative toxicities instead of absolute measures. Finally, we discuss how to avoid pitfalls by distinguishing between unspecific forms of cytotoxicity and specific developmental neurotoxicity. A compilation of compound lists corresponding to the above-discussed principles supplement this review.

1,25-dihydroxyvitamin D3 treatment delays cellular aging in human mesenchymal stem cells while maintaining their multipotent capacity.

1,25-dihydroxyvitamin D3 (1,25D3) was reported to induce premature organismal aging in fibroblast growth factor-23 (Fgf23) and klotho deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC). Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, ß-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence- and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to ß-galactosidase staining and P16 expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105) and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging.

Salicylideneamino-2-thiophenol enhances osteogenic differentiation through the activation of MAPK pathways in multipotent bone marrow stem cell.

Osteoporosis is a reduction in skeletal mass due to an imbalance between bone formation and bone resorption. Therefore, the identification of specific stimulators of bone formation is of therapeutic significance in the treatment of osteoporosis. Salicylideneamino-2-thiophenol (Sal-2) consists of two benzene rings, has been reported to possess antioxidant activity, and is an effective remedy for fever and rheumatic diseases. However, until now the effects of osteoblastic bone formation by Sal-2 were unknown. In this study, we investigated the effects of Sal-2 on osteogenic differentiation of multipotent bone marrow stromal stem cells by alizarin red S staining for osteogenic differentiation, RT-PCR and western blot for alkaline phosphatase (ALP) activity and signaling pathways, FACS analysis and immunofluorescence staining for CD44 and CD51 expression, calcium assays, and immunofluorescence staining for signaling pathways. We found that Sal-2 enhanced the osteogenic differentiation of multipotent bone marrow stromal stem cells. Sal-2 treatment induced the expression and activity of ALP, and enhanced the levels of CD44 and CD51 expression as well as Ca2+ content, in multipotent bone marrow stromal stem cells. Moreover, we found that Sal-2-induced osteogenic differentiation and expression of osteogenesis-related molecules involve the activation of the MAPK and nuclear factor-κB pathways. Our findings provide insight into both the mechanism and effects of Sal-2 on osteogenic differentiation and demonstrate that Sal-2 may be a beneficial adjuvant in stimulating bone formation in osteoporotic diseases.

Impact of bladder-derived acellular matrix, growth factors, and extracellular matrix constituents on the survival and multipotency of marrow-derived mesenchymal stem cells.

We investigate the effect of bladder-derived acellular matrix (ACM) on bone marrow mesenchymal stem cells (BM-MSC) growth, survival, and differentiation, and evaluate the effect of collagen I and IV on BM-MSC differentiation potential to SMC. BM-MSCs isolated from CD1(_) mice were characterized by surface markers and differentiation into different lineages. BM-MSC SMC potential was further evaluated in stem cell medium alone or supplemented with TGF-ß1 and recombinant human platelet-derived growth factor (PDGF-BB) on plastic, collagen I and IV using western blot. Furthermore, BM-MSCs were seeded on porcine derived ACM-fortified with hyaluronic acid and cultured in Mesencult+-growth factors, bone, or fat induction media for 3 weeks. Seeded constructs were evaluated by H&E, Ki67 assay, Oil red O, and Alizarin red stain. SMC differentiation was semiquantified via immunohistochemistry. BM-MSCs differentiated into fat and bone when induced. In Mesencult, BM-MSCs differentiated into SMC, expressing α-SMA, calponin, and MHC. BM-MSCs cultured on collagen I and IV reduced expression of SMC and MHC compared to plastic. On ACM-HA, BM-MSCs maintained multipotent state by differentiating to bone and fat when induced. In Mesencult, BM-MSC-seeded ACM-HA expressed α-SMA, calponin, and MHC. TGF-ß1 and PDGF-BB enhanced SMC differentiation on collagens and ACM-HA. SMC proteins expression by BM-MSC varies depending on culture substrate. SMC markers are expressed higher on plastic and lower on collagen I, IV, and ACM-HA, suggesting these substrates preferentially maintain undifferentiated state of BM-MSC, which could be advantageous for incorporation of cell-seeded grafts to permit host modulation of tissue regeneration.

Synergism of human amnion-derived multipotent progenitor (AMP) cells and a collagen scaffold in promoting brain wound recovery: pre-clinical studies in an experimental model of penetrating ballistic-like brain injury.

One of the histopathological consequences of a penetrating ballistic brain injury is the formation of a permanent cavity. In a previous study using the penetrating ballistic-like brain injury (PBBI) model, engrafted human amnion-derived multipotent progenitor (AMP) cells failed to survive when injected directly in the injury tract, suggesting that the cell survival requires a supportive matrix. In this study, we seated AMP cells in a collagen-based scaffold, injected into the injury core, and investigated cell survival and neuroprotection following PBBI. AMP cells suspended in AMP cell conditioned medium (ACCS) or in a liquefied collagen matrix were injected immediately after a PBBI along the penetrating injury tract. Injured control rats received only liquefied collagen matrix. All animals were allowed to survive two weeks. Consistent with our previous results, AMP cells suspended in ACCS failed to survive; likewise, no collagen was identified at the injury site when injected alone. In contrast, both AMP cells and the collagen were preserved in the injury cavity when injected together. In addition, AMP cells/collagen treatment preserved some apparent brain tissue in the injury cavity, and there was measurable infiltration of endogenous neural progenitor cells and astrocytes into the preserved brain tissue. AMP cells were also found to have migrated into the subventricular zone and the corpus callosum. Moreover, the AMP cell/collagen treatment significantly attenuated the PBBI-induced axonal degeneration in the corpus callosum and ipsilateral thalamus and improved motor impairment on rotarod performance. Overall, collagen-based scaffold provided a supportive matrix for AMP cell survival, migration, and neuroprotection.

[Xenogeneic protein free cultivation of mesenchymal multipotent stromal cells]. / Kultivace mezenchymových kmenových bunek bez xenogenních bílkovin.

PURPOSE OF THE STUDY: The aim of this study was to compare the standard laboratory method of cultivation of mesenchymal multipotent stromal cells (MSC) and a novel technique of rapid MSC expansion focused on simple clinical use. MATERIAL AND METHODS: Bone marrow mononuclear cells of donors were cultured for 14 days by the standard and the new cultivation method. The standard method (STD) was based on an alpha MEM medium supplemented with foetal calf serum (FCS). The new animal protein-free method (CLI) was based on the clinical grade medium CellgroTM, pooled human serum and human recombinant growth factors (EGF, PDGF-BB, M-CSF, FGF-2) supplemented with dexamethasone, insulin and ascorbic acid. The cell product was analyzed by flow cytometry. Furthermore, the cell products of STD and CLI methods were differentiated in vitro, and histochemical and immunohistochemical analyses, electron microscopy and elemental analysis were performed. Some cells were seeded on biodegradable scaffolds, in vivo implanted into immunodeficient mice for 6 weeks and evaluated by histological methods. RESULTS: Yields of the CLI method after 14 days of cultivation were 40-fold higher than those obtained by the STD technique (p<0.05). Cell products of both STD and CLI methods fulfilled the criteria of MSC in terms of antigen expression assessed by flow cytometry, as well as osteogenic, chondrogenic and adipogenic in vitro differentiation assays. Moreover, these cells seeded on three-dimensional scaffolds cultured in osteogenic medium produced mineral deposits and a fibrillar extracellular matrix seen with the electron microscope. Deposits examined by element analysis contained calcium and phosphorus at a ratio of 5 to 3, which corresponded to hydroxyapatite. The cell product seeded on biodegradable scaffolds and implanted into immunodeficient mice was able to form a bone-like calcified tissue with blood supply of mouse origin. DISCUSSION: The currently used methods of cultivation have certain disadvantages compared to the CLI technique, such as a longer cultivation period, need of primary expansion and reseeding and use of FCS with all its potential risks. High yields of cells obtained by the CLI method in a very short time make the use of cultured cells potentially suitable for an acute trauma management. Other therapeutic non-orthotopic applications of CLI-cultured cells have to be further investigated. CONCLUSIONS: The CLI method is unique, rapid, simple and lacking the addition of animal proteins. CLI-cultured cells fulfil the criteria of MSC. The CLI method potentially allows for closed system cultivation in good manufacturing practice (GMP) conditions. It seems to be easily transferable to good clinical practice compared to other protocols and should extend the possibilities of cell therapy and tissue engineering of cartilage and bone. The new method is protected by Czech patent 301 148 and by Europian patent EP 1999250 according to Czech and international laws.