Characterization and response to inflammatory stimulation of human endometrial-derived mesenchymal stem/stromal cells.

BACKGROUND AIMS: The human endometrium has emerged as an attractive source of endometrial-derived mesenchymal stem/stromal cells (eMSCs) that can be easily isolated by non-invasive procedures. The prominent capacity of the endometrium for efficient and scarless regeneration each menstrual cycle indicates the increased eMSC immunomodulatory and pro-angiogenic properties. Herein the authors investigated the molecular responses of eMSCs to an inflammatory environment and whether those intrinsic responses affected their functional attributes. METHODS: Human eMSCs immunophenotypic, transcriptional and secretory profiles were evaluated at passage three (P3) and passage eight (P8) to determine culture effects. Functionally, P3 and P8 non-induced and TNF-α/IFN-γ-induced eMSCs were interrogated for their capacity to suppress stimulated peripheral blood mononuclear cell (PBMC) proliferation, whereas non-induced eMSCs were assessed for their support to vascular network formation in co-cultures with human umbilical vein endothelial cells in vitro. RESULTS: Non-induced P3 and P8 eMSCs exhibited similar spindle-shaped morphology and clonogenic capacity. Nevertheless, P8 eMSCs showed reduced growth rate capacity and telomere length. The eMSCs displayed the typical MSC-related immunophenotypic profile, with P3 and P8 eMSCs expressing high levels (>98%) of CD140ß, intermediate levels (35-60%) of CD146 and SUSD2 and low levels (∼8%) of NG2 pericytic markers. Non-induced P3 and P8 showed similar transcriptional and secretory profiles, though the expression of immunomodulatory HLA-G and IL-8 genes was significantly downregulated in P8 compared with P3 eMSCs. Upon TNF-α/IFN-γ induction, eMSCs showed an immunophenotypic profile similar to that of non-induced eMSCs, except for significant upregulation of HLA-DR protein expression in both induced P3 and P8 eMSCs. However, induced P3 and P8 eMSCs showed significant upregulation of CD10, HLA-G, IDO, IL-6, IL-8, LIF and TSG gene expression compared with non-induced cultures. TNF-α/IFN-γ induction strongly increased the secretion of inflammatory-/angiogenesis-related molecules, whereas growth factor secretion was similar to the non-induced eMSCs. Functionally, P3 and P8 eMSCs showed a strong inhibitory effect on stimulated PBMC proliferation and the capacity to support neovascularization in vitro. CONCLUSIONS: The authors' study suggests that serial expansion does not affect eMSC immunophenotypic, transcriptional and secretory profiles. This is directly reflected by the functional immunomodulatory and pro-angiogenic properties of eMSCs, which remain unaltered until P8 in vitro. However, exposure of eMSCs to inflammatory environments enhances their immunomodulatory transcriptional and inflammatory-/angiogenesis-related secretory profiles. Therefore, the resulting evidence of eMSCs serial expansion and exposure to inflammation could serve as a foundation for improved eMSCs manufacturing and potential clinical translation efforts.

Signature quality attributes of CD146+ mesenchymal stem/stromal cells correlate with high therapeutic and secretory potency.

CD146+ bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) play key roles in the perivascular niche, skeletogenesis, and hematopoietic support; however, comprehensive evaluation of therapeutic potency has yet to be determined. In this study, in vitro inflammatory priming to crude human BM-MSCs (n = 8) captured a baseline of signature responses, including enriched CD146+ with coexpression of CD107aHigh , CXCR4High , and LepRHigh , transcriptional profile, enhanced secretory capacity, and robust immunomodulatory secretome and function, including immunopotency assays (IPAs) with stimulated immune cells. These signatures were significantly more pronounced in CD146+ (POS)-sorted subpopulation than in the CD146- (NEG). Mechanistically, POS BM-MSCs showed a markedly higher secretory capacity with significantly greater immunomodulatory and anti-inflammatory protein production upon inflammatory priming compared with the NEG BM-MSCs. Moreover, IPAs with stimulated peripheral blood mononuclear cells and T lymphocytes demonstrated robust immunosuppression mediated by POS BM-MSC while inducing significant frequencies of regulatory T cells. in vivo evidence showed that POS BM-MSC treatment promoted pronounced M1-to-M2 macrophage polarization, ameliorating inflammation/fibrosis of knee synovium and fat pad, unlike treatment with NEG BM-MSCs. These data correlate the expression of CD146 with innately higher immunomodulatory and secretory capacity, and thus therapeutic potency. This high-content, reproducible evidence suggests that the CD146+ (POS) MSC subpopulation are the mediators of the beneficial effects achieved using crude BM-MSCs, leading to translational implications for improving cell therapy and manufacturing.

Therapeutic Potential of Adipose Stem Cells.

Adipose stem cells (ASCs) have gained attention in the fields of stem cells regenerative medicine due to their multifaceted therapeutic capabilities. Promising preclinical evidence of ASCs has supported the substantial interest in the use of these cells as therapy for human disease. ASCs are an adult stem cell resident in adipose tissue with the potential to differentiation along mesenchymal lineages. They also are known to be recruited to sites of inflammation where they exhibit strong immunomodulatory capabilities to promote wound healing and regeneration. ASCs can be isolated from adipose tissue at a relatively high yield compared to their mesenchymal cell counterparts: bone marrow-derived mesenchymal stem cells (BM-MSCs). Like BM-MSCs, ASCs are easily culture expanded and have a reduced immunogenicity or are perhaps immune privileged, making them attractive options for cellular therapy. Additionally, the heterogeneous cellular product obtained after digestion of adipose tissue, called the stromal vascular fraction (SVF), contains ASCs and several populations of stromal and immune cells. Both the SVF and culture expanded ASCs have the potential to be therapeutic in various diseases. This review will focus on the preclinical and clinical evidence of SVF and ASCs, which make them potential candidates for therapy in regenerative medicine and inflammatory disease processes.

Regulatory-compliant conditions during cell product manufacturing enhance in vitro immunomodulatory properties of infrapatellar fat pad-derived mesenchymal stem/stromal cells.

BACKGROUND AIMS: Mesenchymal stem/stromal cell (MSC)-based therapies have gained attention as potential alternatives for multiple musculoskeletal indications based on their trophic and immunomodulatory properties. The infrapatellar fat pad (IFP) serves as a reservoir of MSCs, which play crucial roles modulating inflammatory and fibrotic events at the IFP and its neighboring tissue, the synovium. In an effort to comply with the existing regulatory framework regarding cell-based product manufacturing, we interrogated the in vitro immunomodulatory capacity of human-derived IFP-MSCs processed under different conditions, including a regulatory-compliant protocol, in addition to their response to the inflammatory and fibrotic environments often present in joint disease. METHODS: Immunophenotype, telomere length, transcriptional and secretory immunomodulatory profiles and functional immunopotency assay were assessed in IFP-MSCs expanded in regular fetal bovine serum (FBS)-supplemented medium and side-by-side compared with same-donor cells processed with two media alternatives (i.e., regulatory-compliant pooled human platelet lysate [hPL] and a chemically reinforced/serum-reduced [Ch-R] formulation). Finally, to assess the effects of such formulations on the ability of the cells to respond to pro-inflammatory and pro-fibrotic conditions, all three groups were stimulated ex vivo (i.e., cell priming) with a cocktail containing TNFα, IFNγ and connective tissue growth factor (tumor-initiating cells) and compared with non-induced cohorts assessing the same outcomes. RESULTS: Non-induced and primed IFP-MSCs expanded in either hPL or Ch-R showed distinct morphology in vitro, similar telomere dynamics and distinct phenotypical and molecular profiles when compared with cohorts grown in FBS. Gene expression of IL-8, CD10 and granulocyte colony-stimulating factor was highly enriched in similarly processed IFP-MSCs. Cell surface markers related to the immunomodulatory capacity, including CD146 and CD10, were highly expressed, and secretion of immunomodulatory and pro-angiogenic factors was significantly enhanced with both hPL and Ch-R formulations. Upon priming, the immunomodulatory phenotype was enhanced, resulting in further increase in CD146 and CD10, significant CXCR4 presence and reduction in TLR3. Similarly, transcriptional and secretory profiles were enriched and more pronounced in IFP-MSCs expanded in either hPL or Ch-R, suggesting a synergistic effect between these formulations and inflammatory/fibrotic priming conditions. Collectively, increased indoleamine-2,3-dioxygenase activity and prostaglandin E2 secretion for hPL- and Ch-R-expanded IFP-MSCs were functionally reflected by their robust T-cell proliferation suppression capacity in vitro compared with IFP-MSCs expanded in FBS, even after priming. CONCLUSIONS: Compared with processing using an FBS-supplemented medium, processing IFP-MSCs with either hPL or Ch-R similarly enhances their immunomodulatory properties, which are further increased after exposure to an inflammatory/fibrotic priming environment. This evidence supports the adoption of regulatory-compliant practices during the manufacturing of a cell-based product based on IFP-MSCs and anticipates a further enhanced response once the cells face the pathological environment after intra-articular administration. Mechanistically, the resulting functionally enhanced cell-based product has potential utilization as a novel, minimally invasive cell therapy for joint disease through modulation of local immune and inflammatory events.

Leptin produced by obesity-altered adipose stem cells promotes metastasis but not tumorigenesis of triple-negative breast cancer in orthotopic xenograft and patient-derived xenograft models.

BACKGROUND: Breast cancer is the second leading cause of cancer deaths in the USA. Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer with high rates of metastasis, tumor recurrence, and resistance to therapeutics. Obesity, defined by a high body mass index (BMI), is an established risk factor for breast cancer. Women with a high BMI have increased incidence and mortality of breast cancer; however, the mechanisms(s) by which obesity promotes tumor progression are not well understood. METHODS: In this study, obesity-altered adipose stem cells (obASCs) were used to evaluate obesity-mediated effects of TNBC. Both in vitro and in vivo analyses of TNBC cell lines were co-cultured with six pooled donors of obASCs (BMI > 30) or ASCs isolated from lean women (lnASCs) (BMI < 25). RESULTS: We found that obASCs promote a pro-metastatic phenotype by upregulating genes associated with epithelial-to-mesenchymal transition and promoting migration in vitro. We confirmed our findings using a TNBC patient-derived xenograft (PDX) model. PDX tumors grown in the presence of obASCS in SCID/beige mice had increased circulating HLA1+ human cells as well as increased numbers of CD44+CD24- cancer stem cells in the peripheral blood. Exposure of the TNBC PDX to obASCs also increased the formation of metastases. The knockdown of leptin expression in obASCs suppressed the pro-metastatic effects of obASCs. CONCLUSIONS: Leptin signaling is a potential mechanism through which obASCs promote metastasis of TNBC in both in vitro and in vivo analyses.

Drug resistance profiling of a new triple negative breast cancer patient-derived xenograft model.

BACKGROUND: Triple-negative breast cancer (TNBC) represents an aggressive subtype with limited therapeutic options. Experimental preclinical models that recapitulate their tumors of origin can accelerate target identification, thereby potentially improving therapeutic efficacy. Patient-derived xenografts (PDXs), due to their genomic and transcriptomic fidelity to the tumors from which they are derived, are poised to improve the preclinical testing of drug-target combinations in translational models. Despite the previous development of breast and TNBC PDX models, those derived from patients with demonstrated health-disparities are lacking. METHODS: We use an aggressive TNBC PDX model propagated in SCID/Beige mice that was established from an African-American woman, TU-BcX-2 K1, and assess its metastatic potential and drug sensitivities under distinct in vitro conditions. Cellular derivatives of the primary tumor or the PDX were grown in 2D culture conditions or grown in mammospheres 3D culture. Flow cytometry and fluorescence staining was used to quantify cancer stem cell-like populations. qRT-PCR was used to describe the mesenchymal gene signature of the tumor. The sensitivity of TU-BcX-2 K1-derived cells to anti-neoplastic oncology drugs was compared in adherent cells and mammospheres. Drug response was evaluated using a live/dead staining kit and crystal violet staining. RESULTS: TU-BcX-2 K1 has a low propensity for metastasis, reflects a mesenchymal state, and contains a large burden of cancer stem cells. We show that TU-BcX-2 K1 cells have differential responses to cytotoxic and targeted therapies in 2D compared to 3D culture conditions insofar as several drug classes conferred sensitivity in 2D but not in 3D culture, or cells grown as mammospheres. CONCLUSIONS: Here we introduce a new TNBC PDX model and demonstrate the differences in evaluating drug sensitivity in adherent cells compared to mammosphere, or suspension, culture.

A novel patient-derived xenograft model for claudin-low triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) subtypes are clinically aggressive and cannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derived xenograft methods. METHODS: We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. RESULTS: Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole and intact breast tumor, specific cell populations within the tumor, and the extracellular matrix. CONCLUSIONS: Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.

Adipose stromal vascular fraction attenuates TH1 cell-mediated pathology in a model of multiple sclerosis.

BACKGROUND: The therapeutic efficacy of adipose-derived stem cells (ASCs) has been investigated for numerous clinical indications, including autoimmune and neurodegenerative diseases. Less is known using the crude adipose product called stromal vascular fraction (SVF) as therapy, although our previous studies demonstrated greater efficacy at late-stage disease compared to ASCs in the experimental autoimmune encephalomyelitis (EAE) mouse, a model of multiple sclerosis. In this study, SVF cells and ASCs were administered during the pathogenic progression, designated as early disease, to elucidate immunomodulatory mechanisms when high immune cell activities associated with autoimmune signaling occur. These implications are essential for clinical translation when considering timing of administration for cell therapies. METHODS: We investigated the effects of SVF cells and ASCs by analyzing the spleens, peripheral blood, and central nervous system tissues throughout the course of EAE disease following administration of SVF cells, ASCs, or vehicle. In vitro, immunomodulatory potentials of SVF cells and ASCs were measured when exposed to EAE-derived splenocytes. RESULTS: Interestingly, treatment with SVF cells and ASCs transiently enhanced the severity of disease directly after administration, substantiating this critical immunomodulatory signaling. More importantly, it was only the EAE mice treated with SVF cells that were able to overcome the advancing pathogenesis and showed improvements by the end of the study. The frequency of lesions in spinal cords following SVF treatment correlated with diminished activities of the T helper type 1 cells, known effector cells of this disease. Co-cultures with splenocytes isolated from EAE mice revealed transcripts of interleukin-10 and transforming growth factor-ß, known promoters of regulatory T cells, that were greatly expressed in SVF cells compared to ASCs, and expression levels of signaling mediators related to effector T cells were insignificant in both SVF cells and ASCs. CONCLUSION: This is the first evidence, to date, to elucidate a mechanism of action of SVF treatment in an inflammatory, autoimmune disease. Our data supports key immunomodulatory signaling between cell therapies and T cells in this T cell-mediated disease. Together, treatment with SVF mediated immunomodulatory effects that diminished effector cell activities, promoted regulatory T cells, and reduced neuroinflammation.

Immunomodulatory Effects of Adipose Stromal Vascular Fraction Cells Promote Alternative Activation Macrophages to Repair Tissue Damage.

The pathogenesis of many diseases is driven by the interactions between helper T (TH ) cells and macrophages. The phenotypes of these cells are functional dichotomies that are persuaded according to the surrounding milieu. In both multiple sclerosis and the experimental autoimmune encephalomyelitis (EAE) model, TH 1 and TH 17 cells propagate autoimmune signaling and inflammation in the peripheral lymphoid tissues. In turn, this proinflammatory repertoire promotes the classical activation, formerly the M1-type, macrophages. Together, these cells infiltrate into the central nervous system (CNS) tissues and generate inflammatory and demyelinating lesions. Our most recent report demonstrated the immunomodulatory and anti-inflammatory effects of adipose stromal vascular fraction (SVF) cells and adipose-derived stem cells (ASCs) that led to functional, immunological, and pathological improvements in the EAE model. Here, a deeper investigation revealed the induction of regulatory T cells and alternative activation, or M2-type, macrophages in the periphery followed by the presence of alternative activation macrophages, reduced cellular infiltrates, and attenuation of neuroinflammation in CNS tissues following intraperitoneal administration of these treatments. Spleens from treated EAE mice revealed diminished TH 1 and TH 17 cell activities and were markedly higher in the levels of anti-inflammatory cytokine interleukin-10. Interestingly, SVF cells were more effective than ASCs at mediating these beneficial changes, which were attributed to their localization to the spleens after administration. Together, SVF cells rapidly and robustly attenuated the propagation of autoimmune signaling in the periphery that provided a permissive milieu in the CNS for repair and possibly regeneration. Stem Cells 2017;35:2198-2207.

Adipose Stromal Vascular Fraction-Mediated Improvements at Late-Stage Disease in a Murine Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a common neurodegenerative disease and remains an unmet clinical challenge. In MS, an autoimmune response leads to immune cell infiltration, inflammation, demyelination, and lesions in central nervous system (CNS) tissues resulting in tremors, fatigue, and progressive loss of motor function. These pathologic hallmarks are effectively reproduced in the murine experimental autoimmune encephalomyelitis (EAE) model. The stromal vascular fraction (SVF) of adipose tissue is composed of adipose-derived stromal/stem cells (ASC), adipocytes, and various leukocytes. The SVF can be culture expanded to generate ASC lines. Clinical trials continue to demonstrate the safety and efficacy of ASC therapies for treating several diseases. However, little is known about the effectiveness of the SVF for neurodegenerative diseases, such as MS. At late-stage disease, EAE mice show severe motor impairment. The goal for these studies was to test the effectiveness of SVF cells and ASC in EAE mice after the onset of neuropathology. The clinical scoring, behavior, motor function, and histopathologic analyses revealed significant improvements in EAE mice treated with the SVF or ASC. Moreover, SVF treatment mediated more robust improvements to CNS pathology than ASC treatment based on significant modulations of inflammatory factors. The most pronounced changes following SVF treatment were the high levels of interleukin-10 in the peripheral blood, lymphoid and CNS tissues along with the induction of regulatory T cells in the lymph nodes which indicate potent immunomodulatory effects. The data indicate SVF cells effectively ameliorated the EAE immunopathogenesis and supports the potential use of SVF for treating MS. Stem Cells 2017;35:532-544.

Human Adipose Stromal/Stem Cells from Obese Donors Show Reduced Efficacy in Halting Disease Progression in the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis.

Multiple sclerosis is an autoimmune disease that affects the white matter of the central nervous system and involves inflammation and demyelination. The recent advances in our understanding of adipose-derived stromal/stem cells (ASCs) and the utilization of these cells in clinical settings to treat diseases have made it essential to identify the most effective ASCs for therapy. Studies have not yet investigated the impact of obesity on the therapeutic efficacy of ASCs. Obesity is characterized by adipocyte hyperplasia and hypertrophy and can extend to metabolic and endocrine dysfunction. Investigating the impact obesity has on ASC biology will determine whether these cells are suitable for use in regenerative medicine. The therapeutic efficacy of ASCs isolated from lean subjects (body mass index [BMI] < 25; lnASCs) and obese subjects (BMI > 30; obASCs) were determined in murine experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Compared with the EAE disease-modifying effects of lnASCs, obASCs consistently failed to alleviate clinical symptoms or inhibit inflammation in the central nervous system. When activated, obASCs expressed higher mRNA levels of several pro-inflammatory cytokines compared with lnASCs. Additionally, conditioned media (CM) collected from the obASCs markedly enhanced the proliferation and differentiation of T cells; whereas, CM from lnASC did not. These results indicate that obesity reduces, or eliminates, the anti-inflammatory effects of human ASCs such that they may not be a suitable cell source for the treatment of autoimmune diseases. The data suggest that donor demographics may be particularly important when identifying suitable stem cells for treatment.

Obesity inhibits the osteogenic differentiation of human adipose-derived stem cells.

BACKGROUND: Craniomaxillofacial defects secondary to trauma, tumor resection, or congenital malformations are frequent unmet challenges, due to suboptimal alloplastic options and limited autologous tissues such as bone. Significant advances have been made in the application of adipose-derived stem/stromal cells (ASCs) in the pre-clinical and clinical settings as a cell source for tissue engineering approaches. To fully realize the translational potential of ASCs, the identification of optimal donors for ASCs will ensure the successful implementation of these cells for tissue engineering approaches. In the current study, the impact of obesity on the osteogenic differentiation of ASCs was investigated. METHODS: ASCs isolated from lean donors (body mass index <25; lnASCs) and obese donors (body mass index >30; obASCs) were induced with osteogenic differentiation medium as monolayers in an estrogen-depleted culture system and on three-dimensional scaffolds. Critical size calvarial defects were generated in male nude mice and treated with scaffolds implanted with lnASCs or obASCs. RESULTS: lnASCs demonstrated enhanced osteogenic differentiation in monolayer culture system, on three-dimensional scaffolds, and for the treatment of calvarial defects, whereas obASCs were unable to induce similar levels of osteogenic differentiation in vitro and in vivo. Gene expression analysis of lnASCs and obASCs during osteogenic differentiation demonstrated higher levels of osteogenic genes in lnASCs compared to obASCs. CONCLUSION: Collectively, these results indicate that obesity reduces the osteogenic differentiation capacity of ASCs such that they may have a limited suitability as a cell source for tissue engineering.

Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers.

INTRODUCTION: The steady increase in the incidence of obesity among adults has been paralleled with higher levels of obesity-associated breast cancer. While recent studies have suggested that adipose stromal/stem cells (ASCs) isolated from obese women enhance tumorigenicity, the mechanism(s) by which this occurs remains undefined. Evidence suggests that increased adiposity results in increased leptin secretion from adipose tissue, which has been shown to increased cancer cell proliferation. Previously, our group demonstrated that ASCs isolated from obese women (obASCs) also express higher levels of leptin relative to ASCs isolated from lean women (lnASCs) and that this obASC-derived leptin may account for enhanced breast cancer cell growth. The current study investigates the impact of inhibiting leptin expression in lnASCs and obASCs on breast cancer cell (BCC) growth and progression. METHODS: Estrogen receptor positive (ER+) BCCs were co-cultured with leptin shRNA lnASCs or leptin shRNA obASCs and changes in the proliferation, migration, invasion, and gene expression of BCCs were investigated. To assess the direct impact of leptin inhibition in obASCs on BCC proliferation, MCF7 cells were injected alone or mixed with control shRNA obASCs or leptin shRNA obASCs into SCID/beige mice. RESULTS: ER+ BCCs were responsive to obASCs during direct co-culture, whereas lnASCs were unable to increase ER(+) BCC growth. shRNA silencing of leptin in obASCs negated the enhanced proliferative effects of obASC on BCCs following direct co-culture. BCCs co-cultured with obASCs demonstrated enhanced expression of epithelial-to-mesenchymal transition (EMT) and metastasis genes (SERPINE1, MMP-2, and IL-6), while BCCs co-cultured with leptin shRNA obASCs did not display similar levels of gene induction. Knockdown of leptin significantly reduced tumor volume and decreased the number of metastatic lesions to the lung and liver. These results correlated with reduced expression of both SERPINE1 and MMP-2 in tumors formed with MCF7 cells mixed with leptin shRNA obASCs, when compared to tumors formed with MCF7 cells mixed with control shRNA obASCs. CONCLUSION: This study provides mechanistic insight as to how obesity enhances the proliferation and metastasis of breast cancer cells; specifically, obASC-derived leptin contributes to the aggressiveness of breast cancer in obese women.

Multipotent stromal cells alleviate inflammation, neuropathology, and symptoms associated with globoid cell leukodystrophy in the twitcher mouse.

Globoid cell leukodystrophy (GLD) is a common neurodegenerative lysosomal storage disorder caused by a deficiency in galactocerebrosidase (GALC), an enzyme that cleaves galactocerebroside during myelination. Bone marrow transplantation has shown promise when administered to late-onset GLD patients. However, the side effects (e.g., graft vs. host disease), harsh conditioning regimens (e.g., myelosuppression), and variable therapeutic effects make this an unsuitable option for infantile GLD patients. We previously reported modest improvements in the twitcher mouse model of GLD after intracerebroventricular (ICV) injections of a low-dose of multipotent stromal cells (MSCs). Goals of this study were to improve bone marrow-derived MSC (BMSC) therapy for GLD by increasing the cell dosage and comparing cell type (e.g., transduced vs. native), treatment timing (e.g., single vs. weekly), and administration route (e.g., ICV vs. intraperitoneal [IP]). Neonatal twitcher mice received (a) 2 × 10(5) BMSCs by ICV injection, (b) 1 × 10(6) BMSCs by IP injection, (c) weekly IP injections of 1 × 10(6) BMSCs, or (d) 1 × 10(6) lentiviral-transduced BMSCs overexpressing GALC (GALC-BMSC) by IP injection. All treated mice lived longer than untreated mice. However, the mice receiving peripheral MSC therapy had improved motor function (e.g., hind limb strength and rearing ability), twitching symptoms, and weight compared to both the untreated and ICV-treated mice. Inflammatory cell, globoid cell, and apoptotic cell levels in the sciatic nerves were significantly decreased as a result of the GALC-BMSC or weekly IP injections. The results of this study indicate a promising future for peripheral MSC therapy as a noninvasive, adjunct therapy for patients affected with GLD.

Correction to: A Chemically Defined Common Medium for Culture of C2C12 Skeletal Muscle and Human Induced Pluripotent Stem Cell Derived Spinal Spheroids.

[This corrects the article DOI: 10.1007/s12195-020-00624-1.].

Human-derived osteoblast-like cells and pericyte-like cells induce distinct metastatic phenotypes in primary breast cancer cells.

Approximately 70% of advanced breast cancer patients will develop bone metastases, which accounts for ∼90% of cancer-related mortality. Breast cancer circulating tumor cells (CTCs) establish metastatic tumors in the bone after a close interaction with local bone marrow cells including pericytes and osteoblasts, both related to resident mesenchymal stem/stromal cells (BM-MSCs) progenitors. In vitro recapitulation of the critical cellular players of the bone microenvironment and infiltrating CTCs could provide new insights into their cross-talk during the metastatic cascade, helping in the development of novel therapeutic strategies. Human BM-MSCs were isolated and fractionated according to CD146 presence. CD146+ cells were utilized as pericyte-like cells (PLCs) given the high expression of the marker in perivascular cells, while CD146- cells were induced into an osteogenic phenotype generating osteoblast-like cells (OLCs). Transwell migration assays were performed to establish whether primary breast cancer cells (3384T) were attracted to OLC. Furthermore, proliferation of 3384T breast cancer cells was assessed in the presence of PLC- and OLC-derived conditioned media. Additionally, conditioned media cultures as well as transwell co-cultures of each OLCs and PLCs were performed with 3384T breast cancer cells for gene expression interrogation assessing their induced transcriptional changes with an emphasis on metastatic potential. PLC as well as their conditioned media increased motility and invasion potential of 3384T breast cancer cells, while OLC induced a dormant phenotype, downregulating invasiveness markers related with migration and proliferation. Altogether, these results indicate that PLC distinctively drive 3384T cancer cells to an invasive and migratory phenotype, while OLC induce a quiescence state, thus recapitulating the different phases of the in vivo bone metastatic process. These data show that phenotypic responses from metastasizing cancer cells are influenced by neighboring cells at the bone metastatic niche during the establishment of secondary metastatic tumors.

Human Tendon Stem/Progenitor Cell Features and Functionality Are Highly Influenced by in vitro Culture Conditions.

Our understanding of tendon biology continues to evolve, thus leading to opportunities for developing novel, evidence-based effective therapies for the treatment of tendon disorders. Implementing the knowledge of tendon stem/progenitor cells (TSPCs) and assessing their potential in enhancing tendon repair could fill an important gap in this regard. We described different molecular and phenotypic profiles of TSPCs modulated by culture density, as well as their multipotency and secretory activities. Moreover, in the same experimental setting, we evaluated for different responses to inflammatory stimuli mediated by TNFα and IFNγ. We also preliminarily investigated their immunomodulatory activity and their role in regulating degradation of substance P. Our findings indicated that TSPCs cultured at low density (LD) exhibited cobblestone morphology and a reduced propensity to differentiate. A distinctive immunophenotypic profile was also observed with high secretory and promising immunomodulatory responses when primed with TNFα and IFNγ. In contrast, TSPCs cultured at high density (HD) showed a more elongated fibroblast-like morphology, a greater adipogenic differentiation potential, and a higher expression of tendon-related genes with respect to LD. Finally, HD TSPCs showed immunomodulatory potential when primed with TNFα and IFNγ, which was slightly lower than that shown by LD. A shift from low to high culture density during TSPC expansion demonstrated intermediate features confirming the cellular adaptability of TSPCs. Taken together, these experiments allowed us to identify relevant differences in TSPCs based on culture conditions. This ability of TSPCs to acquire distinguished morphology, phenotype, gene expression profile, and functional response advances our current understanding of tendons at a cellular level and suggests responsivity to cues in their in situ microenvironment.

Molecular Crosstalk Between Macrophages and Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) have been widely investigated for regenerative medicine applications, from treating various inflammatory diseases as a cell therapy to generating engineered tissue constructs. Numerous studies have evaluated the potential effects of MSCs following therapeutic administration. By responding to their surrounding microenvironment, MSCs may mediate immunomodulatory effects through various mechanisms that directly (i.e., contact-dependent) or indirectly (i.e., paracrine activity) alter the physiology of endogenous cells in various disease pathologies. More specifically, a pivotal crosstalk between MSCs and tissue-resident macrophages and monocytes (TMφ) has been elucidated using in vitro and in vivo preclinical studies. An improved understanding of this crosstalk could help elucidate potential mechanisms of action (MOAs) of therapeutically administered MSCs. TMφ, by nature of their remarkable functional plasticity and prevalence within the body, are uniquely positioned as critical modulators of the immune system - not only in maintaining homeostasis but also during pathogenesis. This has prompted further exploration into the cellular and molecular alterations to TMφ mediated by MSCs. In vitro assays and in vivo preclinical trials have identified key interactions mediated by MSCs that polarize the responses of TMφ from a pro-inflammatory (i.e., classical activation) to a more anti-inflammatory/reparative (i.e., alternative activation) phenotype and function. In this review, we describe physiological and pathological TMφ functions in response to various stimuli and discuss the evidence that suggest specific mechanisms through which MSCs may modulate TMφ phenotypes and functions, including paracrine interactions (e.g., secretome and extracellular vesicles), nanotube-mediated intercellular exchange, bioenergetics, and engulfment by macrophages. Continued efforts to elucidate this pivotal crosstalk may offer an improved understanding of the immunomodulatory capacity of MSCs and inform the development and testing of potential MOAs to support the therapeutic use of MSCs and MSC-derived products in various diseases.

CD10/Neprilysin Enrichment in Infrapatellar Fat Pad-Derived Mesenchymal Stem Cells Under Regulatory-Compliant Conditions: Implications for Efficient Synovitis and Fat Pad Fibrosis Reversal.

BACKGROUND: Synovitis and infrapatellar fat pad (IFP) fibrosis participate in various conditions of the knee. Substance P (SP), a neurotransmitter secreted within those structures and historically associated with nociception, also modulates local neurogenic inflammatory and fibrotic responses. Exposure of IFP mesenchymal stem cells (IFP-MSCs) to a proinflammatory/profibrotic environment (ex vivo priming with TNFα, IFNγ, and CTGF) induces their expression of CD10/neprilysin, effectively degrading SP in vitro and in vivo. PURPOSE/HYPOTHESIS: The purpose was to test the therapeutic effects of IFP-MSCs processed under regulatory-compliant protocols, comparing them side-by-side with standard fetal bovine serum (FBS)-grown cells. The hypothesis was that when processed under such protocols, IFP-MSCs do not require ex vivo priming to acquire a CD10-rich phenotype efficiently degrading SP and reversing synovitis and IFP fibrosis. STUDY DESIGN: Controlled laboratory study. METHODS: Human IFP-MSCs were processed in FBS or either of 2 alternative conditions-regulatory-compliant pooled human platelet lysate (hPL) and chemically reinforced medium (Ch-R)-and then subjected to proinflammatory/profibrotic priming with TNFα, IFNγ, and CTGF. Cells were assessed for in vitro proliferation, stemness, immunophenotype, differentiation potential, transcriptional and secretory profiles, and SP degradation. Based on a rat model of acute synovitis and IFP fibrosis, the in vivo efficacy of cells degrading SP plus reversing structural signs of inflammation and fibrosis was assessed. RESULTS: When compared with FBS, IFP-MSCs processed with either hPL or Ch-R exhibited a CD10High phenotype and showed enhanced proliferation, differentiation, and immunomodulatory transcriptional and secretory profiles (amplified by priming). Both methods recapitulated and augmented the secretion of growth factors seen with FBS plus priming, with some differences between them. Functionally, in vitro SP degradation was more efficient in hPL and Ch-R, confirmed upon intra-articular injection in vivo where CD10-rich IFP-MSCs also dramatically reversed signs of synovitis and IFP fibrosis even without priming or at significantly lower cell doses. CONCLUSION: hPL and Ch-R formulations can effectively replace FBS plus priming to induce specific therapeutic attributes in IFP-MSCs. The resulting fine-tuned, regulatory-compliant, cell-based product has potential future utilization as a novel minimally invasive cell therapy for the treatment of synovitis and IFP fibrosis. CLINICAL RELEVANCE: The therapeutic enhancement of IFP-MSCs manufactured under regulatory-compliant conditions suggests that such a strategy could accelerate the time from preclinical to clinical phases. The therapeutic efficacy obtained at lower MSC numbers than currently needed and the avoidance of cell priming for efficient results could have a significant effect on the design of clinical protocols to potentially treat conditions involving synovitis and IFP fibrosis.

A Chemically Defined Common Medium for Culture of C2C12 Skeletal Muscle and Human Induced Pluripotent Stem Cell Derived Spinal Spheroids.

INTRODUCTION: Multicellular platforms and linked multi organ on chip devices are powerful tools for drug discovery, and basic mechanistic studies. Often, a critical constraint is defining a culture medium optimal for all cells present in the system. In this study, we focused on the key cells of the neuromuscular junction i.e., skeletal muscle and motor neurons. METHODS: Formulation of a chemically defined medium for the co-culture of C2C12 skeletal muscle cells and human induced pluripotent stem cell (hiPSC) derived spinal spheroids (SpS) was optimized. C2C12 cells in 10 experimental media conditions and 2 topographies were evaluated over a 14-day maturation period to determine the ideal medium formulation for skeletal muscle tissue development. RESULTS: During early maturation, overexpression of genes for myogenesis and myopathy was observed for several media conditions, corresponding to muscle delamination and death. Together, we identified 3 media formulations that allowed for more controlled differentiation, healthier muscle tissue, and long-term culture duration. This evidence was then used to select media formulations to culture SpS and subsequently assessed axonal growth. As axonal growth in SpS cultures was comparable in all selected media conditions, our data suggest that the neuronal basal medium with no added supplements is the ideal medium formulation for both cell types. CONCLUSIONS: Optimization using both topographical cues and culture media formulations provides a comprehensive analyses of culture conditions that are vital to future applications for in vitro NMJ models.