Xenogenic Application of Human Placenta-Derived Mesenchymal Stromal Cells in a Porcine Large Animal Model.

In animal models, cell therapies for different diseases or injuries have been very successful. Preclinical studies with cells aiming at a stroke, heart attack, and other emergency situations were promising but sometimes failed translation in clinical situations. We, therefore, investigated if human placenta-derived mesenchymal stromal cells can be injected in pigs without provoking rejection to serve as a xenogenic transplantation model to bridge preclinical animal studies to more promising future preclinical studies. Male human placenta-derived mesenchymal stromal cells were isolated, expanded, and characterized by flow cytometry, in vitro differentiation, and quantitative reverse-transcription polymerase chain reaction to prove their nature. Such cells were injected into the sphincter muscle of the urethrae of female pigs under visual control by cystoscopy employing a Williams needle. The animals were observed over 7 days of follow-up. Reactions of the host to the xenogeneic cells were explored by monitoring body temperature, and inflammatory markers including IL-1ß, CRP, and haptoglobin in blood. After sacrifice on day 7, infiltration of inflammatory cells in the tissue targeted was investigated by histology and immunofluorescence. DNA of injected human cells was detected by PCR. Upon injection in vascularized porcine tissue, human placenta-derived mesenchymal stromal cells were tolerated, and systemic inflammatory parameters were not elevated. DNA of injected cells was detected in situ 7 days after injection, and moderate local infiltration of inflammatory cells was observed. The therapeutic potential of human placenta-derived mesenchymal stromal cells can be explored in porcine large animal models of injury or disease. This seems a promising strategy to explore technologies for cell injections in infarcted hearts or small organs and tissues in therapeutically relevant amounts requiring large animal models to yield meaningful outcomes.

Production of Proliferation- and Differentiation-Competent Porcine Myoblasts for Preclinical Studies in a Porcine Large Animal Model of Muscular Insufficiency.

Muscular insufficiency is observed in many conditions after injury, chronic inflammation, and especially in elderly populations. Causative cell therapies for muscle deficiencies are not state of the art. Animal models to study the therapy efficacy are, therefore, needed. We developed an improved protocol to produce myoblasts suitable for pre-clinical muscle therapy studies in a large animal model. Myoblasts were isolated from the striated muscle, expanded by employing five different protocols, and characterized on transcript and protein expression levels to determine procedures that yielded optimized regeneration-competent myoblasts and multi-nucleated myotubes. We report that swine skeletal myoblasts proliferated well under improved conditions without signs of cellular senescence, and expressed significant levels of myogenic markers including Pax7, MyoD1, Myf5, MyoG, Des, Myf6, CD56 (p ≤ 0.05 each). Upon terminal differentiation, myoblasts ceased proliferation and generated multi-nucleated myotubes. Injection of such myoblasts into the urethral sphincter complex of pigs with sphincter muscle insufficiency yielded an enhanced functional regeneration of this muscle (81.54% of initial level) when compared to the spontaneous regeneration in the sham controls without myoblast injection (67.03% of initial level). We conclude that the optimized production of porcine myoblasts yields cells that seem suitable for preclinical studies of cell therapy in a porcine large animal model of muscle insufficiency.

Regulation of Immune Checkpoint Antigen CD276 (B7-H3) on Human Placenta-Derived Mesenchymal Stromal Cells in GMP-Compliant Cell Culture Media.

Therapies utilizing autologous mesenchymal cell delivery are being investigated as anti-inflammatory and regenerative treatments for a broad spectrum of age-related diseases, as well as various chronic and acute pathological conditions. Easily available allogeneic full-term human placenta mesenchymal stromal cells (pMSCs) were used as a potential pro-regenerative, cell-based therapy in degenerative diseases, which could be applied also to elderly individuals. To explore the potential of allogeneic pMSCs transplantation for pro-regenerative applications, such cells were isolated from five different term-placentas, obtained from the dissected maternal, endometrial (mpMSCs), and fetal chorion tissues (fpMSCs), respectively. The proliferation rate of the cells in the culture, as well as their shape, in vitro differentiation potential, and the expression of mesenchymal lineage and stem cell markers, were investigated. Moreover, we studied the expression of immune checkpoint antigen CD276 as a possible modulation of the rejection of transplanted non-HLA-matched homologous or even xeno-transplanted pMSCs. The expression of the cell surface markers was also explored in parallel in the cryosections of the relevant intact placenta tissue samples. The expansion of pMSCs in a clinical-grade medium complemented with 5% human platelet lysate and 5% human serum induced a significant expression of CD276 when compared to mpMSCs expanded in a commercial medium. We suggest that the expansion of mpMSCs, especially in a medium containing platelet lysate, elevated the expression of the immune-regulatory cell surface marker CD276. This may contribute to the immune tolerance towards allogeneic pMSC transplantations in clinical situations and even in xenogenic animal models of human diseases. The endurance of the injected comparably young human-term pMSCs may promote prolonged effects in clinical applications employing non-HLA-matched allogeneic cell therapy for various degenerative disorders, especially in aged adults.

Elevated Expression of the Immune Checkpoint Ligand CD276 (B7-H3) in Urothelial Carcinoma Cell Lines Correlates Negatively with the Cell Proliferation.

The cell surface molecule CD276 (B7-H3) is an immune checkpoint antigen. The elevated expression of CD276 on tumors contributes to the suppression of anti-tumor T-cell responses and correlates with poor prognosis. METHODS: The expression of CD276 was explored in vitro on eight urothelial carcinoma cell lines (UM-UC) in comparison to eight normal urothelial cells (NUCs) by RT-qPCR, Western blotting, and flow cytometry. Cell proliferation was enumerated over consecutive passages. The expression of cancer stem cell markers CD24 and CD44, cytokeratins, and vimentin was investigated by immunofluorescence. The expression of CD276 in bladder tumor samples and metastases was explored by immunohistochemistry. RESULTS: Expression of CD276 on cell surfaces was elevated on UM-UCs when compared to NUCs. In UM-UCs, CD276 transcripts correlated moderately positive with CD276 protein expression (ρ = 0.660) and strongly positive with CD276 surface-expression (ρ = 0.810). CD276 mRNA expression (ρ = -0.475) and CD276 protein expression (ρ = -0.417) had a significant negative correlation with proliferation, while a significant correlation between proliferation and cell surface expression was not observed in UM-UCs. CONCLUSION: The expression of CD276 on UM-UC bladder tumor cell surfaces is elevated. Slow proliferating UM-UC cells express more CD276 mRNA and protein than fast proliferating cells. In patients, slow proliferating CD276high tumor (stem) cells may evade immune surveillance. However, cancer therapy targeting CD276 may be effective in the treatment of slow proliferating tumor cells.

Replacing Needle Injection by a Novel Waterjet Technology Grants Improved Muscle Cell Delivery in Target Tissues.

Current regimen to treat patients suffering from stress urinary incontinence often seems not to yield satisfactory improvement or may come with severe side effects. To overcome these hurdles, preclinical studies and clinical feasibility studies explored the potential of cell therapies successfully and raised high hopes for better outcome. However, other studies were rather disappointing. We therefore developed a novel cell injection technology to deliver viable cells in the urethral sphincter complex by waterjet instead of using injection needles. We hypothesized that the risk of tissue injury and loss of cells could be reduced by a needle-free injection technology. Muscle-derived cells were obtained from young male piglets and characterized. Upon expansion and fluorescent labeling, cells were injected into cadaveric tissue samples by either waterjet or injection needle. In other experiments, labeled cells were injected by waterjet in the urethra of living pigs and incubated for up to 7 days of follow-up. The analyses documented that the cells injected by waterjet in vitro were viable and proliferated well. Upon injection in live animals, cells appeared undamaged, showed defined cellular somata with distinct nuclei, and contained intact chromosomal DNA. Most importantly, by in vivo waterjet injections, a significantly wider cell distribution was observed when compared with needle injections (P < .05, n ≥ 12 samples). The success rates of waterjet cell application in living animals were significantly higher (≥95%, n = 24) when compared with needle injections, and the injection depth of cells in the urethra could be adapted to the need by adjusting waterjet pressures. We conclude that the novel waterjet technology injects viable muscle cells in tissues at distinct and predetermined depth depending on the injection pressure employed. After waterjet injection, loss of cells by full penetration or injury of the tissue targeted was reduced significantly in comparison with our previous studies employing needle injections.

Injection of Porcine Adipose Tissue-Derived Stroma Cells via Waterjet Technology.

Urinary incontinence (UI) is a highly prevalent condition characterized by the deficiency of the urethral sphincter muscle. Regenerative medicine branches, particularly cell therapy, are novel approaches to improve and restore the urethral sphincter function. Even though injection of active functional cells is routinely performed in clinical settings by needle and syringe, these approaches have significant disadvantages and limitations. In this context, needle-free waterjet (WJ) technology is a feasible and innovative method that can inject viable cells by visual guided cystoscopy in the urethral sphincter. In the present study, we used WJ to deliver porcine adipose tissue-derived stromal cells (pADSCs) into cadaveric urethral tissue and subsequently investigated the effect of WJ delivery on cell yield and viability. We also assessed the biomechanical features (i.e., elasticity) by atomic force microscopy (AFM) measurements. We showed that WJ delivered pADSCs were significantly reduced in their cellular elasticity. The viability was significantly lower compared to controls but is still above 80%.

Expression of CD146 and Regenerative Cytokines by Human Placenta-Derived Mesenchymal Stromal Cells upon Expansion in Different GMP-Compliant Media.

Mesenchymal stromal cells (MSCs) have been successfully employed in clinical applications. In most studies, autologous MSCs from the bone marrow (bmMSCs) were used, and others employed autologous adipose tissue-derived stromal cells (ADSCs). Recently, clinical feasibility studies provided evidence that MSCs from human term placenta (pMSCs) can be used for homologous therapy facilitating access to regenerative cells in emergency situations, when autologous cells are not available or not suitable. We therefore investigated the expression of MSC stemness marker CD146 and the expression of neuro- and myoregenerative cytokines by human pMSCs after expansion in three different media compliant with good manufacturing protocols (GMP) in comparison to pMSCs expanded in a commercial MSC expansion media. To replace xenobiotic serum in the GMP-compliant media employed in this study, either human serum, human serum plus platelet lysate (PLL), or human plasma plus PLL was used. We report that enrichment of media with PLL accelerates pMSC proliferation but reduces the expression of the stemness marker CD146 significantly, while PLL deprivation enhanced the CD146 expression. In contrast, the reduced expression of CD146 by PLL deprivation was not observed on bmMSCs. The expression of the cytokines investigated was not modulated significantly by PLL. We conclude that accelerated expansion of pMSCs in GMP-compliant media enriched by PLL reduces the expression of stemness marker CD146, but does not influence the expression of neuro- and myoregenerative cytokines.

Injection of Porcine Adipose Tissue-Derived Stromal Cells by a Novel Waterjet Technology.

Previously, we developed a novel, needle-free waterjet (WJ) technology capable of injecting viable cells by visual guided cystoscopy in the urethral sphincter. In the present study, we aimed to investigate the effect of WJ technology on cell viability, surface markers, differentiation and attachment capabilities, and biomechanical features. Porcine adipose tissue-derived stromal cells (pADSCs) were isolated, expanded, and injected by WJ technology. Cell attachment assays were employed to investigate cell-matrix interactions. Cell surface molecules were analyzed by flow cytometry. Cells injected by Williams Needle (WN), normal cannula, or not injected cells served as controls. Biomechanical properties were assessed by atomic force microscopy (AFM). pADSCs injected by the WJ were viable (85.9%), proliferated well, and maintained their in vitro adipogenic and osteogenic differentiation capacities. The attachment of pADSCs was not affected by WJ injection and no major changes were noted for cell surface markers. AFM measurements yielded a significant reduction of cellular stiffness after WJ injections (p < 0.001). WJ cell delivery satisfies several key considerations required in a clinical context, including the fast, simple, and reproducible delivery of viable cells. However, the optimization of the WJ device may be necessary to further reduce the effects on the biomechanical properties of cells.

Rapid and precise delivery of cells in the urethral sphincter complex by a novel needle-free waterjet technology.

OBJECTIVES: To investigate the therapy of stress urinary incontinence in a preclinical setting cells were injected into the urethrae of minipigs; however, cells injected by William's needle were frequently misplaced or lost; thus, we investigated if needle-free cell injections using a novel waterjet technology facilitates precise injections in the urethral sphincter complex. MATERIALS AND METHODS: Porcine adipose tissue-derived stromal cells (pADSCs) were isolated from boars, expanded, labelled, and injected in the sphincter of female pigs by waterjet employing two different protocols. After incubation for 15 min or 3 days, the urethrae of the pigs were examined. Injected cells were visualised by imaging and fluorescence microscopy of tissue sections. DNA of injected male cells was verified by polymerase chain reaction (PCR) of the sex-determining region (SRY) gene. Cell injections by William's needle served as controls. RESULTS: The new waterjet technology delivered pADSCs faster and with better on-site precision than the needle injections. Bleeding during or after waterjet injection or other adverse effects, such as swelling or urinary retention, were not observed. Morphologically intact pADSCs were detected in the urethrae of all pigs treated by waterjet. SRY-PCR of chromosomal DNA and detection of recombinant green fluorescent protein verified the injection of viable cells. In contrast, three of four pigs injected by William's needle displayed no or misplaced cells. CONCLUSION: Transurethral injection of viable pADSCs by waterjet is a simple, fast, precise, and yet gentle new technology. This is the first proof-of-principle concept study providing evidence that a waterjet injects intact cells exactly in the tissue targeted in a preclinical in vivo situation. To further explore the clinical potential of the waterjet technology longer follow-up, as well as incontinence models have to be studied.

A novel waterjet technology for transurethral cystoscopic injection of viable cells in the urethral sphincter complex.

AIMS: In a recent preclinical study, we noticed that injection of cells in the urethral sphincter by needle through a cystoscope under visual control frequently yielded in misplacement or loss of cells. We, therefore, investigated if a needle-free waterjet device delivers viable cells under defined settings, including injection volume and pressure, fluid velocity and transportation media, precisely through the urothelium and connective tissue close to the sphincter muscle without full penetration of the sphincter apparatus. METHODS: Mesenchymal stromal cells (MSCs) were prepared for needle-free waterjet injections. Upon injections into liquids cell viability and yield were investigated by trypan blue dye exclusion. Upon injection into cadaveric urethral tissue samples, cells were isolated from the urethrae and expanded to prove that this novel method delivered viable cells into the tissue. MSC injections by William's needle served as controls. RESULTS: Waterjet injections of MSCs into isotonic cell culture medium resulted in equal or better yields of viable cells when compared with needle injections. Upon injection in urethral tissue samples, the waterjet technology facilitated fast and precise injections of viable cells through urothelial, mucosal and submucosal layers to reach the sphincter muscle. By controlling the injection pressure, loss of cells due to insufficient thrust or unintended full penetration was avoided. CONCLUSIONS: Needle-free waterjet injections deliver cells in the urethra faster and more precisely when compared with needle injections without compromising their viability. This is the first proof-of-concept study providing evidence that a waterjet transports viable cells precisely into the targeted tissue.

Choice of xenogenic-free expansion media significantly influences the myogenic differentiation potential of human bone marrow-derived mesenchymal stromal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have great potential for use in cell-based therapies for restoration of structure and function of many tissue types including smooth muscle. METHODS: We compared proliferation, immunophenotype, differentiation capability and gene expression of bone marrow-derived MSCs expanded in different media containing human serum, plasma and platelet lysate in combination with commonly used protocols for myogenic, osteogenic, chondrogenic and adipogenic differentiation. Moreover, we developed a xenogenic-free protocol for myogenic differentiation of MSCs. RESULTS: Expansion of MSCs in media complemented with serum, serum + platelet lysate or plasma + platelet lysate were multipotent because they differentiated toward four mesenchymal (myogenic, osteogenic, chondrogenic, adipogenic) lineages. Addition of platelet lysate to expansion media increased the proliferation of MSCs and their expression of CD146. Incubation of MSCs in medium containing human serum or plasma plus 5% human platelet lysate in combination with smooth muscle cell (SMC)-inducing growth factors TGFß1, PDGF and ascorbic acid induced high expression of ACTA2, TAGLN, CNN1 and/or MYH11 contractile SMC markers. Osteogenic, adipogenic and chondrogenic differentiations served as controls. DISCUSSION: Our study provides novel data on the myogenic differentiation potential of human MSCs toward the SMC lineage using different xenogenic-free cell culture expansion media in combination with distinct differentiation medium compositions. We show that the choice of expansion medium significantly influences the differentiation potential of human MSCs toward the smooth muscle cell, as well as osteogenic, adipogenic and chondrogenic lineages. These results can aid in designing studies using MSCs for tissue-specific therapeutic applications.

Bone marrow-derived mesenchymal stromal cells differ in their attachment to fibronectin-derived peptides from term placenta-derived mesenchymal stromal cells.

INTRODUCTION: Human mesenchymal stromal cells (MSCs) can be isolated from different sources including bone marrow and term placenta. These two populations display distinct patterns of proliferation and differentiation in vitro. Since proliferation and differentiation of cells are modulated by cell-matrix interactions, we investigated the attachment of MSCs to a set of peptide-coated surfaces and explored their interactions with peptides in suspension. METHODS: Human MSCs were isolated from bone marrow and term placenta and expanded. Binding of MSCs to peptides was investigated by a cell-attachment spot assay, by blocking experiments and flow cytometry. The integrin expression pattern was explored by a transcript array and corroborated by quantitative reverse transcription polymerase chain reaction and flow cytometry. RESULTS: Expanded placenta-derived MSCs (pMSCs) attached well to surfaces coated with fibronectin-derived peptides P7, P15, and P17, whereas bone marrow-derived MSCs (bmMSCs) attached to P7, but barely to P15 and P17. The binding of bmMSCs and pMSCs to the peptides was mediated by ß1 integrins. In suspension, expanded bmMSCs barely bind to P7, P13, P15, and less to P14 and P17. Ex vivo, bmMSCs failed to bind P7, but displayed a weak interaction with P13, P14, and P15. In suspension, expanded pMSCs displayed binding to many peptides, including P4, P7, P13, P14, P15, and P17. The differences observed in binding of bmMSCs and pMSCs to the peptides were associated with significant differences in expression of integrin α2-, α4-, and α6-chains. CONCLUSIONS: Human bmMSCs and pMSCs show distinct patterns of attachment to defined peptides and maintain differences in expression of integrins in vitro. Interactions of ex vivo bmMSCs with a given peptide yield different staining patterns compared to expanded bmMSCs in suspension. Attachment of expanded MSCs to peptides on surfaces is different from interactions of expanded MSCs with peptides in suspension. Studies designed to investigate the interactions of human MSCs with peptide-augmented scaffolds or peptides in suspension must therefore regard these differences in cell-peptide interactions.

Human Placenta-Derived CD146-Positive Mesenchymal Stromal Cells Display a Distinct Osteogenic Differentiation Potential.

Mesenchymal stromal cells (MSCs) are multipotent cells that can be differentiated in vitro into a variety of cell types, including adipocytes or osteoblasts. Our recent studies indicated that a high expression of CD146 on MSCs from bone marrow correlates with their robust osteogenic differentiation potential. We therefore investigated if expression of CD146 on MSCs from the placenta correlates with a similar osteogenic differentiation potential. The MSCs were isolated specifically from the endometrial and fetal parts of human term placenta and expanded in separate cultures and compared with MSCs from bone marrow as controls. The expression of cell surface antigens was investigated by flow cytometry. Differentiation of MSCs was documented by cytochemistry and analysis of typical lineage marker genes. CD146-positive MSCs were separated from CD146-negative cells by magnet-assisted cell sorts (MACS). We report that the expression of CD146 is associated with a higher osteogenic differentiation potential in human placenta-derived MSCs (pMSCs) and the CD146(pos) pMSCs generated a mineralized extracellular matrix, whereas the CD146(neg) pMSCs failed to do so. In contrast, adipogenic and chondrogenic differentiation of pMSCs was not different in CD146(pos) compared with CD146(neg) pMSCs. Upon enrichment of pMSCs by MACS, the CD146(neg) and CD146(pos) populations maintained their expression levels for this antigen for several passages in vitro. We conclude that CD146(pos) pMSCs either respond to osteogenic stimuli more vividly or, alternatively, CD146(pos) pMSCs present a pMSC subset that is predetermined to differentiate into osteoblasts.

The active form of leflunomide, HMR1726, facilitates TNF-alpha and IL-17 induced MMP-1 and MMP-3 expression.

BACKGROUND/AIMS: To elucidate the influence and mode of action of HMR1726 (the active metabolite of leflunomide) on TNF-alpha and IL-17 activated metalloproteinases expression in synoviocytes. METHODS: Synovial fibroblasts from RA and OA patients were stimulated with both cytokines and altered gene expression in the presence or absence of leflunomide was detected by microarray analyses and quantitative RT-PCR. Protein expression was detected by western blotting and commercial ELISAs. RESULTS: Microarray analyses revealed that the addition of HMR1726 (50 microM) to TNF-alpha and IL-17- stimulated synoviocytes induced gene expression of metallo-proteinases, especially MMP-1 and -3 in comparison to activated synoviocytes in the absence of leflunomide. To confirm these data, we examined the influence of different concentrations of HMR1726 in synoviocytes from further 5 OA and 7 RA patients by quantitative PCR. HMR1726 gradually induced MMP-1 and MMP-3 gene expression in a dose-dosedependent manner. Similar results were observed on protein levels. Examination of signal transduction pathways participating in the regulation of leflunomideinduced MMPs expression showed that the mechanism underlying activation of MMP-1 is in part p38- and activation of MMP-3 was MEK1/2- dependent. CONCLUSION: Leflunomide was not able to abolish expression of metallo-proteinases in synoviocytes activated with TNF-a and IL-17.