MSC Population Research Articles

Osteogenic potential of heterogeneous and CD271-enriched mesenchymal stromal cells cultured on apatite-wollastonite 3D scaffolds

BackgroundMesenchymal stromal cells (MSCs) are widely used in clinical trials for bone repair and regeneration. Despite previous evidence showing a prominent osteogenic potential of 2D cultured CD271 enriched MSCs, the osteogenic potential of CD271 enriched cells cultured on 3D scaffold is unknown. Apatite-wollastonite glass ceramic (A-W) is an osteoconductive biomaterial shown to be compatible with MSCs. This is the first study comparing the attachment, growth kinetics, and osteogenic potential of two MSC populations, namely heterogeneous plastic adherence MSCs (PA-MSCs) and CD271-enriched MSCs (CD271-MSCs), when cultured on A-W 3D scaffold.ResultsThe paired MSC populations were assessed for their attachment, growth kinetics and ALP activity using confocal and scanning electron microscopy and the quantifications of DNA contents and p-nitrophenyl (pNP) production respectively. While the PA-MSCs and CD271-MSCs had similar expansion and tri-lineage differentiation capacity during standard 2D culture, they showed different proliferation kinetics when seeded on the A-W scaffolds. PA-MSCs displayed a well-spread attachment with more elongated morphology compared to CD271-MSCs, signifying a different level of interaction between the cell populations and the scaffold surface. Following scaffold seeding PA-MSCs fully integrated into the scaffold surface and showed a stronger propensity for osteogenic differentiation as indicated by higher ALP activity than CD271-MSCs. Furthermore, A-W scaffold seeded uncultured non-enriched bone marrow mononuclear cells also demonstrated a higher proliferation rate and greater ALP activity compared to their CD271-enriched counterpart.ConclusionsOur findings suggest that CD271-positive enrichment of a population is not beneficial for osteogenesis when the cells are seeded on A-W scaffold. Furthermore, unselected heterogeneous MSCs or BM-MNCs are more promising for A-W scaffold based bone regeneration. This leads to a conclusion of broader clinical relevance for tissue engineering: on the basis of our observations here the osteogenic potential observed in 2D cell culture should not be considered indicative of likely performance in a 3D scaffold based system, even when one of the cell populations is effectively a subset of the other.

Electroporation: A Sustainable and Cell Biology Preserving Cell Labeling Method for Adipogenous Mesenchymal Stem Cells.

Human mesenchymal stem cells derived from adipose tissue (AD-hMSCs) represent a promising source for tissue engineering and are already widely used in cell therapeutic clinical trials. Until today, an efficient and sustainable cell labeling system for cell tracking does not exist. We evaluated transient transfection through electroporation for cell labeling and compared it with lentiviral transduction for AD-hMSCs. In addition, we tested whether nonsense DNA or a reporter gene such as enhanced green fluorescent protein (EGFP) is the more suitable label for AD-hMSCs. Using electroporation, the transfection efficiency reached a maximal level of 44.6 ± 1.1% EGFP-positive cells after selective and expansive cultivation of the mixed MSC population, and was 44.5 ± 1.4% after gene transfer with Cyanin3-marked nonsense-label DNA, which remained stable during 2 weeks of nonselective cultivation (37.2 ± 4.7% positive AD-hMSCs). Electroporation with both nonsense DNA and pEGFP-N1 led to a slight growth retardation of 45.2% and 59.1%, respectively. EGFP-transfected or transduced AD-hMSCs showed a limited adipogenic and osteogenic differentiation capacity, whereas it was almost unaffected in cells electroporated with the nonsense-label DNA. The nonsense DNA was detectable through quantitative real-time polymerase chain reaction for at least 5 weeks/10 passages and in differentiated AD-hMSCs. EGFP-labeled cells were trackable for 24 h in vitro and served as testing cells with new materials for dental implants for 7 days. In contrast, lentivirally transduced AD-hMSCs showed an altered natural immune phenotype of the AD-hMSCs with lowered expression of two cell type defining surface markers (CD44 and CD73) and a relevantly decreased cell growth by 71.8% as assessed by the number of colony-forming units. We suggest electroporation with nonsense DNA as an efficient and long-lasting labeling method for AD-hMSCs with the comparably lowest negative impact on the phenotype or the differentiation capacity of the cells, which may, therefore, be suitable for tissue engineering. In contrast, EGFP transfection by electroporation is efficient but may be more suitable for cell tracking within cell therapies without MSC differentiation procedures. Since current protocols of lentiviral gene transduction include the risk of cell biological alterations, electroporation seems advantageous and sustainable enough for hMSC labeling.

Molecular and cellular basis of mammary gland fibrosis and cancer risk.

Mammary gland luminal cells are maintained by the proliferation of ER- luminal progenitor (LP) cells. Human breast LP cells exhibit telomere DNA damage, which is associated with mammographic density and increased cancer risk. Telomeric repeat factor 2 (TRF2) protects telomeres from DNA damage response. TRF2 expression is reduced in human breast cancers. We deleted TRF2 expression in mammary gland epithelium. Mammary glands lacking TRF2 expression exhibited increased telomere DNA damage response, histopathological and functional degeneration, and prominent ductal fibrosis. TRF2-deficient mammary tumors exhibited rapid onset and increased proliferation. Tumor derived LP cells failed to form tumors after transplantation. The MSC population was highly tumorigenic and maintained telomeres via the ALT mechanism. Telomere DNA damage response in mammary tumors resulted in p53 dependent ER+ cellular differentiation and sensitivity to anti-estrogen therapy. Our results provide a new in vivo model of mammographic density, stem cell differentiation, cancer risk, and therapeutic sensitivity.

The Effect of Vancomycin on the Viability and Osteogenic Potential of Bone-Derived Mesenchymal Stem Cells.

Traditionally, methicillin-resistant Staphylococcus aureus (MRSA) is treated with vancomycin, administrated intravenously or applied directly onto infected tissue. The effect of direct (as opposed to systemic) vancomycin treatment on bone formation and remodelling is largely unknown. The minimal inhibitory concentration (MIC) of vancomycin was determined by adding 200μL of different concentrations (1-20μg/mL) to actively growing cultures of S. aureus Xen 31 (methicillin-resistant) and S. aureus Xen 36 (methicillin-sensitive), respectively, and recording changes in optical density over 24h. Bone marrow-derived and proximal femur-derived mesenchymal stem cells (bmMSCs and pfMSCs) from rat femora were exposed to 1 × MIC (5μg/mL) and 4 × MIC (20μg/mL) of vancomycin for 7days. Cell viability was determined by staining with crystal violet and MTT (3-(4,5- di methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), respectively, and osteogenic differentiation by staining with Alizarin Red S. Vancomycin had no effect on the viability of bmMSCs and pfMSCs, even at high levels (20μg/mL). The osteogenic differentiation of pfMSCs was partially inhibited, while osteogenesis in bmMSCs was not severely affected. The direct application of vancomycin to infected bone tissue, even at excessive levels, may preserve the viability of resident MSC populations.

Profiling Mesenchymal Stem Cell Immunomodulation In Vitro

Abstract Mesenchymal stem cells (MSCs) are multipotent adult stem cells found in a variety of tissues, including bone marrow, adipose, and umbilical cord. Although MSCs have the ability to differentiate into a variety of cell types useful for regenerative medicine, their ability to modulate the immune system has been at the forefront in the clinic. The mechanisms by which MSCs affect the immune system are not fully understood. This makes it difficult to design specific quality control measures for MSCs used for cell therapy as well as to understand what parameters are the most critical for their immunomodulatory abilities. This specific testing is important to determine batch-to-batch variability and to better forecast patient-to-patient efficacy. In this study, we demonstrate a workflow to investigate the effects of MSCs on immune cell subset populations. Human MSCs were co-cultured with human T cell subtypes (Th1, Th2, Th17, and Treg) that have been differentiated utilizing CellXVivo™ Differentiation Kits. Co-culture supernates are then analyzed via Proteome Profiler™ Antibody Arrays to screen for changes in cytokine levels, relative to MSCs cultured in the absence of T-cells. Analytes discovered to change in the screening arrays are then quantitated using Luminex® Assays. We hypothesize that these tools and techniques will allow for a better understanding of the mechanisms contributing to MSC immunomodulation and provide methods for routine quality control testing of MSC populations prior to clinical use.

FRI-211 - Cell therapy for acute liver injury- in vivo efficacy of mesenchymal stromal cells in toxic and immune-mediated murine hepatitis

The ability of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) to immunomodulate offers therapeutic potential in liver injury but the inherent heterogeneity of unsorted MSC populations may explain varied/reduced function as well as posing regulatory challenges. Thus, we aimed to evaluate the therapeutic potential of purified CD362+ MSC infusion in murine models of acute liver injury. UC-MSCs were injected intravenously into mice injured by single dose of Carbon tetrachloride (CCl4) & OVA-BIL mice. MSC used were either unsorted or sorted CD362+. The extent of liver damage was determined by liver histology, serum analysis, gene expression and FACS analysis 3 or 5 days after cell infusion. Homing and bio-distribution of stem cells was determined by whole mouse cryo-imaging of Q-dot labelled MSC following infusion of UC-MSC into injured mice. CD362+ MSC were as effective as unsorted MSC in ameliorating liver injury, with reductions in serum ALT seen in both models. In contrast heat-inactivated MSC had no effect on liver injury. MSC also led to a reduction in CD45+staining on liver sections in both models of liver injury corroborated by an accompanying reduction in hepatic CD45+ cells in (FACS analysis of liver digest). In addition, there was a significant reduction in hepatic CD19+ B cells in digested liver in CCl4 injury. CD362+ MSCs were found to have the ability to reduce the level of adhesion molecules (ICAM and VCAM) in Ova-Bil mice. Cryo-imaging of time-course in both animal models indicated that MSC had migrated to the lung within 1 hour and were then cleared rapidly, although there was a liverspecific increase in MSC 2-3 day in Ova-Bil mice. CD362+ human MSC exert potent anti-inflammatory activity in toxic and immune-mediated murine liver injury with demonstrable reductions in infiltrating inflammatory leucocytes and B cells.

Pooled human serum: A new culture supplement for bioreactor-based cell therapies. Preliminary results

BackgroundBone Marrow MSCs are an appealing source for several cell-based therapies. Many bioreactors, as the Quantum Cell Expansion System, have been developed to generate a large number of MSCs under Good Manufacturing Practice conditions by using Human Platelet Lysate (HPL). Previously we isolated in the human bone marrow a novel cell population, named Mesodermal Progenitor Cells (MPCs), which we identified as precursors of MSCs. MPCs could represent an important cell source for regenerative medicine applications. As HPL gives rise to a homogeneus MSC population, limiting the harvesting of other cell types, in this study we investigated the efficacy of pooled human AB serum (ABS) to provide clinically relevant numbers of both MSCs and MPCs for regenerative medicine applications by using the Quantum System. MethodsBone marrow aspirates were obtained from healthy adult individuals undergoing routine total hip replacement surgery and used to generate primary cultures in the bioreactor. HPL and ABS were tested as supplements to culture medium. Morphological observations, cytofluorimetric analysis, lactate and glucose level assessment were performed. ResultsABS gave rise to both heterogeneous MSC and MPC population. About 95% of cells cultured in HPL showed a fibroblast-like morphology and typical mesenchymal surface markers, but MPCs were scarcely represented. DiscussionThe use of ABS appeared to sustain a large scale MSC production, as well as the recovery of a subset of MPCs, and resulted a suitable alternative to HPL in the cell generation based on the Quantum System.

Soluble Collagen VI treatment enhances mesenchymal stem cells expansion for engineering cartilage.

Bone Marrow‐derived mesenchymal stem cells (BM‐MSC) are an attractive source for cell‐based therapies in cartilage injury owing to their efficient differentiation into chondrocytes and their immune‐suppressive abilities. However, their clinical use is hampered by a scarcity of cells leading to compromised efficacy. While expansion of human MSC ex vivo can potentially overcome the scarcity of cells, current methods lead to a rapid loss of the stem cell properties. In this study, we report soluble Collagen VI (cartilage pericellular matrix component) as a potential biologic that can expand the MSC population while maintaining the stem cell phenotype as confirmed by expression of the stem cell markers CD105 and CD90. Short‐term treatment with Collagen VI additionally retains the potential of MSC to differentiate into mature chondrocytes in pellet culture. Cartilage pellets generated from MSC treated with Collagen VI or control express comparable amounts of the chondrogenic markers Collagen II, Aggrecan and Sox9, and the extracellular glycosaminoglycans. Our observations confirm that the use of the endogenous and cartilage‐specific factor Collagen VI is valuable for a rapid and efficient expansion of MSC for potential use in cartilage regeneration and osteoarthritis.

Adaptation of a Simple Microfluidic Platform for High-Dimensional Quantitative Morphological Analysis of Human Mesenchymal Stromal Cells on Polystyrene-Based Substrates.

Multipotent stromal cells (MSCs, often called mesenchymal stem cells) have garnered significant attention within the field of regenerative medicine because of their purported ability to differentiate down musculoskeletal lineages. Given the inherent heterogeneity of MSC populations, recent studies have suggested that cell morphology may be indicative of MSC differentiation potential. Toward improving current methods and developing simple yet effective approaches for the morphological evaluation of MSCs, we combined passive pumping microfluidic technology with high-dimensional morphological characterization to produce robust tools for standardized high-throughput analysis. Using ultraviolet (UV) light as a modality for reproducible polystyrene substrate modification, we show that MSCs seeded on microfluidic straight channel devices incorporating UV-exposed substrates exhibited morphological changes that responded accordingly to the degree of substrate modification. Substrate modification also effected greater morphological changes in MSCs seeded at a lower rather than higher density within microfluidic channels. Despite largely comparable trends in morphology, MSCs seeded in microscale as opposed to traditional macroscale platforms displayed much higher sensitivity to changes in substrate properties. In summary, we adapted and qualified microfluidic cell culture platforms comprising simple straight channel arrays as a viable and robust tool for high-throughput quantitative morphological analysis to study cell-material interactions.

Abstract 4778: Telomere DNA damage promotes mammary gland fibrosis and estrogen receptor-positive breast cancer

Abstract Human breast luminal progenitor cells exhibit telomere DNA damage, which is associated with mammographic density and increased cancer risk. Telomeric repeat factor 2 (TRF2) protects telomeres from DNA damage response and its expression is reduced in human ductal carcinoma in situ and breast cancers. To determine the relationships between telomere DNA damage, mammary gland biology, and cancer risk we genetically deleted TRF2 expression in the mouse mammary gland. TRF2 conditional null mutant mice also were bred with tumor prone MMTV-Wnt1 animals. Telomere DNA damage response was determined by localization of 53BP1 at telomeres and activation of ATM/Chk2/p53 signaling by western blot. Mammary gland and tumor histopathology were determined by hematoxylin and eosin staining. Cell proliferation and ER+ differentiation was determined by immunohistochemistry. Tumor derived CD24+/CD49f+ mammary stem cells and CD61+ luminal progenitor cells were transplanted to mammary fat pads of immunodeficient mice. Alternative lengthening of telomeres was determined by PML localization at telomeres, chromosome orientation-fluorescence in situ hybridization, and telomere circular DNA amplification. Tumor bearing mice were injected with 15 mg raloxifene to determine mammary tumor anti-estrogen sensitivity. Mammary glands lacking TRF2 expression exhibited increased telomere DNA damage response, histopathological and functional degeneration, and prominent ductal fibrosis. TRF2 deficient mammary tumors exhibited rapid onset and increased proliferation. Tumor derived LP cells failed to form tumors following transplantation. The MSC population was highly tumorigenic and maintained telomeres via the ALT mechanism. Telomere DNA damage response in mammary tumors resulted in p53 dependent ER+ cellular differentiation and sensitivity to anti-estrogen therapy. Telomere DNA damage correlated with estrogen receptor expression in human breast cancer. We concluded that telomere DNA damage resulted in mammary gland fibrosis, reduced tumor latency, ER+ differentiation, and sensitivity to anti-estrogen therapy. Citation Format: Jianchun Wu, David L. Crowe. Telomere DNA damage promotes mammary gland fibrosis and estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4778. doi:10.1158/1538-7445.AM2017-4778

Abstract AP17: OVARIAN TUMOR STIMULATION CONVERTS NORMAL OVARY AND OMENTAL MESENCHYMAL STEM CELLS INTO TUMOR–PROMOTING CARCINOMA–ASSOCIATED MESENCHYMAL STEM CELLS

Abstract BACKGROUND: Ovarian carcinoma-associated mesenchymal stem cells (CA-MSCs) are multipotent cells which influence the creation of the ovarian tumor microenvironment. CA-MSCs strongly promote ovarian cancer growth, cancer stem cell-like cells and chemotherapy resistance. CA-MSCs lack tumor associated somatic mutations, however compared to normal MSCs, CA-MSCs have a unique expression profile which mediates their pro-tumorigenic properties. The origin of CA-MSCs is unknown. We hypothesized that CA-MSCs are derived from local tissue MSCs reprogrammed by the ovarian tumor to become pro-tumorigenic CA-MSCs. METHODS: We define a “CA-MSC-like expression profile” of 6 genes significantly and reproducibly differentially expressed between normal human MSCs (from bone marrow (BM), omentum (OM) or ovary (OV)) and patient-derived CA-MSCs. Three normal MSC populations were grown with human ovarian cancer cells and analyzed to determine if tumor stimulation can induce a CA-MSC-like expression profile. Tumor stimulated MSCs were functionally analyzed to determine if acquisition of a CA-MSC-like expression profile correlates with development of pro-tumorigenic functions. Methylation array analysis of normal MSCs versus CA-MSCs was performed to explore the role of epigenetic regulation in the formation a CA-MSC. RESULTS: Tumor stimulation of normal OV and OM MSCs but not BM MSCs yield expression changes which approximate the CA-MSC-like expression profile. In vivo tumor stimulation of normal OM MSCs most effectively induces a CA-MSC-like expression profile. The development of a CA-MSC-like expression profile correlates with gain of pro-tumorigenic functions including induction of cancer cell chemotherapy resistance, enrichment of the ovarian cancer stem cell-like pool and enhancement of tumor growth. In contrast, tumor stimulated BM MSCs--which do not develop a CA-MSC-like expression profile--fail to gain pro-tumorigenic functions. Further, methylation array analysis demonstrates significant global differences in methylation patterns of CA-MSCs versus normal MSCs, indicating that epigenetic alterations may drive the development of the CA-MSC phenotype. CONCLUSIONS: Collectively we demonstrate that normal OV and OM MSCs are capable of becoming CA-MSCs through in vivo tumor stimulation with development of a distinct CA-MSC-like expression profile with corresponding pro-tumorigenic functions. These findings highlight the critical influence of the tumor on the normal host environment, manipulating normal cells to become cancer-supporting cells. The differential capacity of OV and OM MSCs but not BM MSCs to become CA-MSCs also highlights the heterogeneity of MSCs and may factor into the intraperitoneal tropism of ovarian cancer spread. Ultimately, understanding how tumor stimulation drives the development of CA-MSCs may identify powerful targets to disrupt the formation and function of the ovarian tumor microenvironment. Citation Format: Coffman, LG and Buckanovich, RJ. OVARIAN TUMOR STIMULATION CONVERTS NORMAL OVARY AND OMENTAL MESENCHYMAL STEM CELLS INTO TUMOR–PROMOTING CARCINOMA–ASSOCIATED MESENCHYMAL STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP17.

In vitro cell surface markers are insufficient to identify in vivo/in situ multipotent synovial mesenchymal stem cells isolated from normal or osteoarthritic knees

Purpose: Osteoarthritis (OA) is a chronic disease that results in the degeneration of the articular cartilage, but also effects all tissues present in the joint. Current treatments for OA patients focus on pain/symptom relief, however, the core persisting issue of cartilage degeneration remains unmodified. Synovial mesenchymal stem cells derived from the synovium (sMSCs), are an attractive source for articular cartilage regeneration since they demonstrate robust chondrogenic capacity. However, it is now becoming accepted that MSC populations can demonstrate significant phenotypically heterogeneity, which in turn, may affect their potential therapeutic efficacy. Furthermore, it remains unknown if this heterogeneity is natural (e.g. multiple MSC sub-populations present) or if MSCs are influenced by factors in vivo (such as the disease stage of the individual) and/or in vitro (such as culture microenvironment). Additionally, the methods used to define MSCs, such as marker profile could be influenced by varying laboratory/cell culture conditions suggesting that cell surface markers present on MSCs in vitro could be distinct from those that are present in vivo. Therefore, it may be possible that in vitro defined MSC markers may be unreliable to use as a standard to isolate MSCs directly from in vivo/in situ which would be necessary to understand and characterize MSC heterogeneity. A more complete understanding of MSC heterogeneity would allow us to identify and purify only sMSCs that have both self-renewal capacity as well as tri-lineage potential. To address this gap in knowledge we re-assessed the cell surface markers in vivo that are widely used to identify and purify MSCs in vitro. To accomplish this aim and also address questions on MSC heterogeneity, we examined the cell surface marker profiles (in vivo/in situ vs. in vitro) between clonal sMSCs isolated from OA and normal individuals. Additionally, we correlated the cell surface profile in situ with the tri-lineage potential to determine if the differentiation potential of sMSCs can be predicted based on their in situ cell surface expression profile. Methods: Synovium tissue was collected from knees of late stage OA patients (n = 15) and normal controls (n = 21). These biopsies were digested with collagenase type IV and the cell suspension was stained for: MSCs markers (CD90, CD44, CD73, CD271, CD105), a macrophage marker (CD68), and a viability marker (FVS510). An unbiased index sorted approach was utilized where 1 cell/well (per 96 well plate) was sorted based on the absence of CD68 and FVS510. Expanded clones were differentiated into adipocytes, chondrocytes and osteoblasts. Differentiation was analyzed via qPCR and histology. Each in vitro expanded clonal population was also re-examined for surface marker expression. Results: Figure 1 displays the differentiation capacity of 4 clones isolated from a single OA patient. Regardless of the in situ marker profiles expression, all sMSC clones, when cultured in vitro, converged to a unified marker profile of: CD90+, CD44+, CD73+ and CD105+. The in situ marker profile that was correlated with multipotent differentiation capacity of sMSCs was found to be similar between OA and normal clones (CD90+, CD44+, and CD73+). A second cohort of patients was recruited to validate the in situ MSC profile, however, it was found that isolation of CD90+, CD44+, and CD73+ triple positive cells didn't guarantee multipotent differentiation potential as tested by qPCR and histology analysis. Conclusions: This study has demonstrated that using in vitro defined MSC markers to select sMSCs populations directly from human synovial tissue is not specific and the markers currently used by the field are not capable of distinguishing MSCs with or without full differentiation capacity if employed in situ/in vivo. Furthermore, after reviewing in situ vs. in vitro surface marker expression data, it was found that all sMSC sub-populations present in situ (based on marker expression) converge to an indiscriminate profile after culturing. Overall, this suggests that at least some of the MSC markers employed are likely artifacts of the cell culturing process. Thus, the results of this project raise a potentially larger question regarding the identification of MSCs with these standard markers. Do these markers truly describe MSCs? This question should be thoroughly addressed before MSC therapies are continued to be delivered to patients suffering from OA.

Abstract P4-09-01: Tumor suppressor control of the cancer stem cell angiogenic niche

Abstract Mammary stem cells (MSC) expansion is associated with aggressive human breast cancer. The nuclear receptor peroxisome proliferator activated receptor γ (PPARγ) is a breast cancer tumor suppressor, but the mechanisms of this suppression are not completely characterized. To determine if PPARγ regulates MSC expansion in mammary cancer, we deleted PPARγ expression in the mammary epithelium of an in vivo model of basal breast cancer. Loss of PPARγ expression reduced tumor latency, and expanded the CD24+/CD49fhi MSC population. PPARγ null mammary tumors exhibited increased angiogenesis which was detected in human breast cancer. In vivo inhibition of a PPARγ regulated miR-15a/angiopoietin-1 pathway blocked increased angiogenesis and MSC expansion. PPARγ bound and activated a canonical response element in the miR-15a gene. PPARγ null tumors were sensitive to the targeted anti-angiogenic drug sunitinib but resistant to cytotoxic chemotherapy. Normalization of tumor vasculature with sunitinib resulted in objective response to cytotoxic chemotherapy. Chemotherapy treated PPARγ null mammary tumors exhibited luminal phenotype and expansion of unipotent CD61+ luminal progenitor cells. Transplantation of chemotherapy treated luminal progenitor cells recapitulated the luminal phenotype. These results have important implications for anti-angiogenic therapy in breast cancer patients. Citation Format: Crowe D, Kramer K, Wu J. Tumor suppressor control of the cancer stem cell angiogenic niche [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-09-01.

β-Catenin Accumulation is Associated with Increased Expression of Nanog Protein and Predicts Maintenance of MSC Self-Renewal

Human mesenchymal stem cells (hMSCs) are self-renewing cells with the ability to differentiate into organized, functional network of cells. Recent studies have revealed that activation of the Wnt/β-catenin pathway by a glycogen synthase kinase (GSK)-3-specific pharmacological inhibitor, Bio, results in the maintenance of self-renewal in both mouse and human ES cells. The molecular mechanism behind the maintenance of hMSCs by these factors, however, is not fully understood. We found that rEGF enhances the level of β-catenin, a component of the Wnt/β-catenin signaling pathway. Furthermore, it was found that β-catenin upregulates Nanog. EGF activates the β-catenin pathway via the Ras protein and also increased the Nanog protein and gene expression levels 2 h after rEGF treatment. These results suggest that adding EGF can enhance β-catenin and Nanog expression in MSCs and facilitate EGF-mediated maintenance of MSC self-renewal. EGF was shown to augment MSC proliferation while preserving early progenitors within MSC population and thus did not induce differentiation. Thus, EGF not only can be used to expand MSC in vitro but also be utilized to autologous transplantation of MSCs in vivo.

Quantitative Assessment of Optimal Bone Marrow Site for the Isolation of Porcine Mesenchymal Stem Cells.

Background. One of the most plentiful sources for MSCs is the bone marrow; however, it is unknown whether MSC yield differs among different bone marrow sites. In this study, we quantified cellular yield and evaluated resident MSC population from five bone marrow sites in the porcine model. In addition, we assessed the feasibility of a commercially available platelet concentrator (Magellan® MAR01™ Arteriocyte Medical Systems, Hopkinton, MA) as a bedside stem cell concentration device. Methods. Analyses of bone marrow aspirate (BMA) and concentrated bone marrow aspirate (cBMA) included bone marrow volume, platelet and nucleated cell yield, colony-forming unit fibroblast (CFU-F) number, flow cytometry, and assessment of differentiation potential. Results. Following processing, the concentration of platelets and nucleated cells significantly increased but was not significantly different between sites. The iliac crest had significantly less bone marrow volume; however, it yielded significantly more CFUs compared to the other bone marrow sites. Culture-expanded cells from all tested sites expressed high levels of MSC surface markers and demonstrated adipogenic and osteogenic differentiation potential. Conclusions. All anatomical bone marrow sites contained MSCs, but the iliac crest was the most abundant source of MSCs. Additionally, the Magellan can function effectively as a bedside stem cell concentrator.

Mesenchymal Stromal Cells, Instigator or Suppressor for the Development of MLL-AF9 Induced Acute Myeloid Leukemia?

It has been reported that bone marrow (BM) hematopoietic niche is critical for the maintenance of normal hematopoiesis and the development of hematopoietic disorders. However, little is known about how the different BM niche components contribute to the initiation and progression of acute myeloid leukemia (AML). To explore the role of BM stromal cells in the development of AML, we here characterized the BM stromal cells in AML mouse model induced by transplantation of mouse BM cellstransduced with MLL-AF9 oncogene. We subdivided the BM stromal cells (CD45-TER119-) into mesenchymal stem cells (MSCs, CD31-CD44-CD51+SCA1+), mesenchymal progenitor cells (MPCs, CD31-CD44-CD51+SCA1-), and endothelial cells (CD31+) in the mice after development of AML by multicolor fluorescent activated cell sorting (FACS) (Qian et al., Mol Cell Biol, 2013). We found significantly increased frequencies of BM MSCs (p < 0.0001), MPCs (p < 0.0001), and endothelial cells (p < 0.0001) in the AML mice, indicating an AML-induced alteration of BM stromal cell composition. The increased frequency of MSCs in AML mouse BM was confirmed by the increased number of colony foming unit-fibroblasts in the AML mice (p < 0.0001). Furthermore, in vitro multi-lineage differentiation assay revealed increased adipogenic and osteogenic differentiation capacities of the BM MSCs in the AML mice. Consistent with the increased osteogenic differentiation capacity of AML MSCs, Runx2 mRNA expression was increased in freshly sorted AML MSCs compared to that in the control mice. In addition, quantitative real time PCR on freshly sorted BM MPCs showed a dramatic downregulation of the genes important in hematopoietic stem cell regulation, including angiopoietin like 1 (Angptl1), C-X-C motif chemokine (Cxcl12), and kit ligand (Kitl) in the AML mice. The altered gene expression might contribute to the AML progression.To specifically determine the contribution of MSCs to AML development, we analyzed the MSC population expressing Early B cell factor 2 (Ebf2) (Qian et al., Mol Cell Biol, 2013) by establishing the AML in Ebf2-Egfp transgenic recipient mice. We detected an increased frequency of Ebf2+ cells in the recipient BM after development of AML (p = 0.0018). In addition, by using the triple transgenic Tg(Ebf2-Egfp x Ebf2-CreER x Rosa26-loxpStoploxp-Tomato) mouse model, where Ebf2+ MSCs and their progenies can be traced by tomato fluorescent protein after tamoxifen treatment, we found that the MSCs and MPCs generated from the Ebf2+ MSCs were also increased in the AML mouse BM (p < 0.05).To further investigate the functional contribution of Ebf2+ MSCs to the AML progression, we took advantage of the transgenic Tg(Ebf2-CreER x Rosa26-loxpStoploxp-DTA) mouse model allowing specific deletion of the Ebf2-expressing MSCs. Strikingly, we observed an accelerated initiation of AML, illustrated by the shorter survival of the MSC depleted recipients (Figure 1), compared to the non-depleted recipients, suggesting that Ebf2+ MSCs could suppress AML progression.Taken together, our data show that BM stromal cell composition and function are altered in the mice with AML and the BM MSCs may act to suppress the progression of the MLL-AF9 induced AML. These findings provide new evidence for the niche contribution to the development of AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Angiogenic and anti-inflammatory properties of micro-fragmented fat tissue and its derived mesenchymal stromal cells.

BackgroundAdipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for advanced cell-based therapies. They are routinely obtained enzymatically from fat lipoaspirate (LP) as SVF, and may undergo prolonged ex vivo expansion, with significant senescence and decline in multipotency. Besides, these techniques have complex regulatory issues, thus incurring in the compelling requirements of GMP guidelines. Hence, availability of a minimally manipulated, autologous adipose tissue would have remarkable biomedical and clinical relevance. For this reason, a new device, named Lipogems® (LG), has been developed. This ready-to-use adipose tissue cell derivate has been shown to have in vivo efficacy upon transplantation for ischemic and inflammatory diseases. To broaden our knowledge, we here investigated the angiogenic and anti-inflammatory properties of LG and its derived MSC (LG-MSCs) population.MethodsHuman LG samples and their LG-MSCs were analyzed by immunohistochemistry for pericyte, endothelial and mesenchymal stromal cell marker expression. Angiogenesis was investigated testing the conditioned media (CM) of LG (LG-CM) and LG-MSCs (LG-MSCs-CM) on cultured endothelial cells (HUVECs), evaluating proliferation, cord formation, and the expression of the adhesion molecules (AM) VCAM-1 and ICAM-1. The macrophage cell line U937 was used to evaluate the anti-inflammatory properties, such as migration, adhesion on HUVECs, and release of RANTES and MCP-1.ResultsOur results indicate that LG contained a very high number of mesenchymal cells expressing NG2 and CD146 (both pericyte markers) together with an abundant microvascular endothelial cell (mEC) population. Substantially, both LG-CM and LG-MSC-CM increased cord formation, inhibited endothelial ICAM-1 and VCAM-1 expression following TNFα stimulation, and slightly improved HUVEC proliferation. The addition of LG-CM and LG-MSC-CM strongly inhibited U937 migration upon stimulation with the chemokine MCP-1, reduced their adhesion on HUVECs and significantly suppressed the release of RANTES and MCP-1.ConclusionsOur data indicate that LG micro-fragmented adipose tissue retains either per se, or in its embedded MSCs content, the capacity to induce vascular stabilization and to inhibit several macrophage functions involved in inflammation.

Characterization of intercellular communication and mitochondrial donation by mesenchymal stromal cells derived from the human lung.

BackgroundBone marrow-derived mesenchymal stromal cells (BM-MSCs) are capable of repairing wounded lung epithelial cells by donating cytoplasmic material and mitochondria. Recently, we characterized two populations of human lung-derived mesenchymal stromal cells isolated from digested parenchymal lung tissue (LT-MSCs) from healthy individuals or from lung transplant recipients’ bronchoalveolar lavage fluid (BAL-MSCs). The aim of this study was to determine whether LT-MSCs and BAL-MSCs are also capable of donating cytoplasmic content and mitochondria to lung epithelial cells.MethodsCytoplasmic and mitochondrial transfer was assessed by co-culturing BEAS2B epithelial cells with Calcein AM or Mitotracker Green FM-labelled MSCs. Transfer was then measured by flow cytometry and validated by fluorescent microscopy. Molecular inhibitors were used to determine the contribution of microtubules/tunnelling nanotubes (TNTs, cytochalasin D), gap junctions (carbenoxolone), connexin-43 (gap26) and microvesicles (dynasore).ResultsF-actin microtubules/TNTs extending from BM-MSCs, LT-MSCs and BAL-MSCs to bronchial epithelial cells formed within 45 minutes of co-culturing cells. Each MSC population transferred a similar volume of cytoplasmic content to epithelial cells. Inhibiting microtubule/TNTs, gap junction formation and microvesicle endocytosis abrogated the transfer of cytoplasmic material from BM-MSCs, LT-MSCs and BAL-MSCs to epithelial cells. In contrast, blocking connexin-43 gap junction formation had no effect on cytoplasmic transfer. All MSC populations donated mitochondria to bronchial epithelial cells with similar efficiency. Mitochondrial transfer was reduced in all co-cultures after microtubule/TNT or endocytosis inhibition. Gap junction formation inhibition reduced mitochondrial transfer in BM-MSC and BAL-MSC co-cultures but had no effect on transfer in LT-MSC co-cultures. Connexin-43 inhibition did not impact mitochondrial transfer. Finally, bronchial epithelial cells were incapable of donating cytoplasmic content or mitochondria to any MSC population.ConclusionSimilar to their bone marrow counterparts, LT-MSCs and BAL-MSCs can donate cytoplasmic content and mitochondria to bronchial epithelial cells via multiple mechanisms. Given that BM-MSCs utilize these mechanisms to mediate the repair of damaged bronchial epithelial cells, both LT-MSCs and BAL-MSCs will probably function similarly.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0354-8) contains supplementary material, which is available to authorized users.

Enhanced neuro-therapeutic potential of Wharton's Jelly–derived mesenchymal stem cells in comparison with bone marrow mesenchymal stem cells culture

Substantial inconsistencies in mesenchymal stem (stromal) cell (MSC) therapy reported in early translational and clinical studies may indicate need for selection of the proper cell population for any particular therapeutic purpose. In the present study we have examined stromal stem cells derived either from umbilical cord Wharton's Jelly (WJ-MSC) or bone marrow (BM-MSC) of adult, healthy donors. The cells characterized in accordance with the International Society for Cellular Therapy (ISCT) indications as well as other phenotypic and functional parameters have been compared under strictly controlled culture conditions. WJ-MSC, in comparison with BM-MSC, exhibited a higher proliferation rate, a greater expansion capability being additionally stimulated under low-oxygen atmosphere, enhanced neurotrophic factors gene expression and spontaneous tendency toward a neural lineage differentiation commitment confirmed by protein and gene marker induction. Our data suggest that WJ-MSC may represent an example of immature-type “pre-MSC,” where a substantial cellular component is embryonic-like, pluripotent derivatives with the default neural-like differentiation. These cells may contribute in different extents to nearly all classical MSC populations adversely correlated with the age of cell donors. Our data suggest that neuro-epithelial markers, like nestin, stage specific embryonic antigens-4 or α-smooth muscle actin expressions, may serve as useful indicators of MSC culture neuro-regeneration–associated potency.

A Mathematical Model for Predicting In Vitro Stem Cell Proliferation Kinetics.

Over 150 active clinical trials of mesenchymal stem cell therapies are underway, testing the efficacy in enhancing solid organ and islet transplant, hematopoetic stem cell transplants or in tissue regenerative strategies with a majority utilizing autologous stem cells. With a number of co-morbidities present in the targeted host, reports have identified adverse effects of these comorbidities such as diabetes, prior radiation, to reduce the ability to sufficiently expand the desired clinical stem cell dose, forcing the recipient to utilize third party donors, carrying the risk of allosensitization. Using mathematical modeling, we developed a method to predict with greater than 90% certainty, the number of cells which would be obtained after full 6-passage ex vivo expansion. Methods: 42 healthy juvenile rhesus macaques, 13 cynomolgus macaques and three human mesenchymal stem cell preparations were ex vivo expanded. A power-law mathematical model was applied to the proliferation data of naïve MSCs in the early days of expansion to predict the fold increase in the stem cell population at a future time point. Based on normal distribution, coefficients in the power law model were determined via the solution of a system of coupled equations. Results: Prospective validation of the predictive model was verified at two different laboratories, (n=7) and (n=6) with healthy macaques. The under-predicting ratios were as follows: at day 20, 8±0.04%, at day 30, 15±0.06%, at day 60, 23±0.13% and at day 90, 39±0.16%, demonstrating a lack of underprediction for determining expanded stem cell doses when inputting day 10 expansion data. Using a confounded data set with nonhuman primates showing comorbidities, the model was also verified in streptozocin-induced diabetic (n=6) and irradiated (n=24) macaques as well as healthy human MSC populations (n=3) with similar predicting power. Conclusion: With early day 10 expansion data, this model consistently predicts the minimum number of MSC obtained over a period of 3-12 weeks in both healthy and diseased hosts. With the ability to predict the numbers of stem cells obtained over time in order to cryopreserve adequate autologous stem cell doses, this method, has the potential to enable autologous therapies for subjects which were previously not considered as viable candidates minimizing the risk of mesenchymal stem cell-based therapies.