Transient naive reprogramming corrects hiPS cells functionally and epigenetically.

Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function1-8. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.

Modelling human blastocysts by reprogramming fibroblasts into iBlastoids.

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development1-4. However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization.

Reprogramming roadmap reveals route to human induced trophoblast stem cells.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development1-6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

Delayed Accumulation of H3K27me3 on Nascent DNA Is Essential for Recruitment of Transcription Factors at Early Stages of Stem Cell Differentiation.

Recruitment of transcription factors (TFs) to repressed genes in euchromatin is essential to activate new transcriptional programs during cell differentiation. However, recruitment of all TFs, including pioneer factors, is impeded by condensed H3K27me3-containing chromatin. Single-cell and gene-specific analyses revealed that, during the first hours of induction of differentiation of mammalian embryonic stem cells (ESCs), accumulation of the repressive histone mark H3K27me3 is delayed after DNA replication, indicative of a decondensed chromatin structure in all regions of the replicating genome. This delay provides a critical "window of opportunity" for recruitment of lineage-specific TFs to DNA. Increasing the levels of post-replicative H3K27me3 or preventing S phase entry inhibited recruitment of new TFs to DNA and significantly blocked cell differentiation. These findings suggest that recruitment of lineage-specifying TFs occurs soon after replication and is facilitated by a decondensed chromatin structure. This insight may explain the developmental plasticity of stem cells and facilitate their exploitation for therapeutic purposes.

Dietary Fiber and Microbiota Metabolite Receptors Enhance Cognition and Alleviate Disease in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder with poorly understood etiology. AD has several similarities with other "Western lifestyle" inflammatory diseases, where the gut microbiome and immune pathways have been associated. Previously, we and others have noted the involvement of metabolite-sensing GPCRs and their ligands, short-chain fatty acids (SCFAs), in protection of numerous Western diseases in mouse models, such as Type I diabetes and hypertension. Depletion of GPR43, GPR41, or GPR109A accelerates disease, whereas high SCFA yielding diets protect in mouse models. Here, we extended the concept that metabolite-sensing receptors and SCFAs may be a more common protective mechanism against Western diseases by studying their role in AD pathogenesis in the 5xFAD mouse model. Both male and female mice were included. Depletion of GPR41 and GPR43 accelerated cognitive decline and impaired adult hippocampal neurogenesis in 5xFAD and WT mice. Lack of fiber/SCFAs accelerated a memory deficit, whereas diets supplemented with high acetate and butyrate (HAMSAB) delayed cognitive decline in 5xFAD mice. Fiber intake impacted on microglial morphology in WT mice and microglial clustering phenotype in 5xFAD mice. Lack of fiber impaired adult hippocampal neurogenesis in both W and AD mice. Finally, maternal dietary fiber intake significantly affects offspring's cognitive functions in 5xFAD mice and microglial transcriptome in both WT and 5xFAD mice, suggesting that SCFAs may exert their effect during pregnancy and lactation. Together, metabolite-sensing GPCRs and SCFAs are essential for protection against AD, and reveal a new strategy for disease prevention.Significance Statement Alzheimer's disease (AD) is one of the most common neurodegenerative diseases; currently, there is no cure for AD. In our study, short-chain fatty acids and metabolite receptors play an important role in cognitive function and pathology in AD mouse model as well as in WT mice. SCFAs also impact on microglia transcriptome, and immune cell recruitment. Out study indicates the potential of specialized diets (supplemented with high acetate and butyrate) releasing high amounts of SCFAs to protect against disease.

Iterative Methylation Leads to 3-Methylchuangxinmycin Production in Actinoplanes tsinanensis CPCC 200056.

A new congener of chuangxinmycin (CM) was identified from Actinoplanes tsinanensis CPCC 200056. Its structure was determined as 3-methylchuangxinmycin (MCM) by 1D and 2D NMR. MCM could be generated in vivo from CM by heterologous expression of the vitamin B12-dependent radical SAM enzyme CxnA/A1 responsible for methylation of 3-demethylchuangxinmycin (DCM) in CM biosynthesis, indicating that CxnA/A1 could perform iterative methylation for MCM production. In vitro assays revealed significant activities of CM, DCM, and MCM against Mycobacterium tuberculosis H37Rv and clinically isolated isoniazid/rifampin-resistant M. tuberculosis, suggesting that CM and its derivatives may have potential for antituberculosis drug development.

Exploiting the preferential phagocytic uptake of nanoparticle-antigen conjugates for the effective treatment of autoimmunity.

Tolerance induction is central to the suppression of autoimmunity. Here, we engineered the preferential uptake of nano-conjugated autoantigens by spleen-resident macrophages to re-introduce self-tolerance and suppress autoimmunity. The brain autoantigen, myelin oligodendrocyte glycoprotein (MOG), was conjugated to 200 or 500 nm silica nanoparticles (SNP) and delivered to the spleen and liver-resident macrophages of experimental autoimmune encephalomyelitis (EAE) mice, used as a model of multiple sclerosis. MOG-SNP conjugates significantly reduced signs of EAE at a very low dose (50 µg) compared to the higher dose (>800 µg) of free-MOG. This was associated with reduced proliferation of splenocytes and pro-inflammatory cytokines secretion, decreased spinal cord inflammation, demyelination and axonal damage. Notably, biodegradable porous SNP showed an enhanced disease suppression assisted by elevated levels of regulatory T cells and programmed-death ligands (PD-L1/2) in splenic and lymph node cells. Our results demonstrate that targeting nano-conjugated autoantigens to tissue-resident macrophages in lymphoid organs can effectively suppress autoimmunity.

Genome-Guided Discovery of Pretilactam from Actinosynnema pretiosum ATCC 31565.

Actinosynnema is a small but well-known genus of actinomycetes for production of ansamitocin, the payload component of antibody-drug conjugates against cancers. However, the secondary metabolite production profile of Actinosynnema pretiosum ATCC 31565, the most famous producer of ansamitocin, has never been fully explored. Our antiSMASH analysis of the genomic DNA of Actinosynnema pretiosum ATCC 31565 revealed a NRPS-PKS gene cluster for polyene macrolactam. The gene cluster is very similar to gene clusters for mirilactam and salinilactam, two 26-membered polyene macrolactams from Actinosynnema mirum and Salinispora tropica, respectively. Guided by this bioinformatics prediction, we characterized a novel 26-membered polyene macrolactam from Actinosynnema pretiosum ATCC 31565 and designated it pretilactam. The structure of pretilactam was elucidated by a comprehensive analysis of HRMS, 1D and 2D-NMR, with absolute configuration of chiral carbons predicted bioinformatically. Pretilactam features a dihydroxy tetrahydropyran moiety, and has a hexaene unit and a diene unit as its polyene system. A preliminary antibacterial assay indicated that pretilactam is inactive against Bacillus subtilis and Candida albicans.

The Drosophila eve insulator Homie promotes eve expression and protects the adjacent gene from repression by polycomb spreading.

Insulators can block the action of enhancers on promoters and the spreading of repressive chromatin, as well as facilitating specific enhancer-promoter interactions. However, recent studies have called into question whether the activities ascribed to insulators in model transgene assays actually reflect their functions in the genome. The Drosophila even skipped (eve) gene is a Polycomb (Pc) domain with a Pc-group response element (PRE) at one end, flanked by an insulator, an arrangement also seen in other genes. Here, we show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of the adjacent gene, TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. When Homie is deleted in the context of a large transgene that mimics both eve and TER94 regulation, TER94 is repressed. This repression depends on the eve PRE. Ubiquitous TER94 expression is "replaced" by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed. Repression of TER94 is attributable to spreading of the eve Pc domain into the TER94 locus, accompanied by an increase in histone H3 trimethylation at lysine 27. Other PREs can functionally replace the eve PRE, and other insulators can block PRE-dependent repression in this context. The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication. Our data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions. Thus, insulator activities in a native context include enhancer blocking and enhancer-promoter facilitation, as well as preventing the spread of repressive chromatin.

Immunosuppressive potential of human amnion epithelial cells in the treatment of experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). In recent years, it has been found that cells such as human amnion epithelial cells (hAECs) have the ability to modulate immune responses in vitro and in vivo and can differentiate into multiple cell lineages. Accordingly, we investigated the immunoregulatory effects of hAECs as a potential therapy in an MS-like disease, EAE (experimental autoimmune encephalomyelitis), in mice. METHODS: Using flow cytometry, the phenotypic profile of hAECs from different donors was assessed. The immunomodulatory properties of hAECs were examined in vitro using antigen-specific and one-way mixed lymphocyte proliferation assays. The therapeutic efficacy of hAECs was examined using a relapsing-remitting model of EAE in NOD/Lt mice. T cell responsiveness, cytokine secretion, T regulatory, and T helper cell phenotype were determined in the peripheral lymphoid organs and CNS of these animals. RESULTS: In vitro, hAECs suppressed both specific and non-specific T cell proliferation, decreased pro-inflammatory cytokine production, and inhibited the activation of stimulated T cells. Furthermore, T cells retained their naïve phenotype when co-cultured with hAECs. In vivo studies revealed that hAECs not only suppressed the development of EAE but also prevented disease relapse in these mice. T cell responses and production of the pro-inflammatory cytokine interleukin (IL)-17A were reduced in hAEC-treated mice, and this was coupled with a significant increase in the number of peripheral T regulatory cells and naïve CD4+ T cells. Furthermore, increased proportions of Th2 cells in the peripheral lymphoid organs and within the CNS were observed. CONCLUSION: The therapeutic effect of hAECs is in part mediated by inducing an anti-inflammatory response within the CNS, demonstrating that hAECs hold promise for the treatment of autoimmune diseases like MS.

Lithium chloride improves the efficiency of induced pluripotent stem cell-derived neurospheres.

Induced pluripotent stem cell (iPSC)-derived neurospheres, which consist mainly of neural progenitors, are considered to be a good source of neural cells for transplantation in regenerative medicine. In this study, we have used lithium chloride, which is known to be a neuroprotective agent, in an iPSC-derived neurosphere model, and examined both the formation rate and size of the neurospheres as well as the proliferative and apoptotic status of their contents. Our results showed that lithium enhanced the formation and the sizes of the iPSC-derived neurospheres, increased the number of Ki67-positive proliferating cells, but reduced the number of the TUNEL-positive apoptotic cells. This increased number of Ki67 proliferating cells was secondary to the decreased apoptosis and not to the stimulation of cell cycle entry, as the expression of the proliferation marker cyclin D1 mRNA did not change after lithium treatment. Altogether, we suggest that lithium enhances the survival of neural progenitors and thus the quality of the iPSC-derived neurospheres, which may strengthen the prospect of using lithium-treated pluripotent cells and their derivatives in a clinical setting.

Gray matter volume abnormalities were associated with sustained attention in unmedicated major depression.

BACKGROUND: Impaired sustained attention seems to be a core feature of depression while the anatomical alteration of brain was widely reported in depression patients. The authors aimed to identify the relationship between anatomical brain changes and sustained attention deficits in unmedicated patients with major depressive disorder (MDD). METHODS: A total of 51 medication-free MDD patients and 51 matched healthy controls (HC) underwent high-resolution structural magnetic resonance imaging scanning, and optimized voxel-based morphometry method was performed to analyze the changes of gray matter volume (GMV). We employed a computerized neurocognitive task from the Cambridge Neuropsychological Tests Automated Battery (CANTAB)--Rapid Visual Information Processing (RVP) task--as a measurement of sustained attention. Based on clinical symptoms, 40 patients who had completed CANTAB-RVP test were divided into MDDa (mild depression patients) and MDDb (severe depression patients) groups. Then the relationships among sustained attention, GMV of different regions and clinical symptoms were explored separately. RESULTS: MDD patients showed significant GMV increase in left posterior cingulate cortex (PCC) and inferior frontal gyrus (IFG) (p<0.001, uncorrected), and significant GMV decrease in medial/superior frontal gyrus (MFG/SFG) and lingual gyrus (p<0.001, uncorrected). Structure-cognition correlation analyses revealed that in MDD patients, GMV alterations of the IFG were significantly correlated with sustained attention as measured by the CANTAB-RVP. CONCLUSIONS: Increased GMV values of IFG were associated with sustained attention which may underlie the pathophysiology of MDD or be part of the cognition circuit. In the severe depression patients, sustained attention deficits were positively correlated with clinical symptoms.

Efficient B cell responses to Borrelia hermsii infection depend on BAFF and BAFFR but not TACI.

T cell-independent antibody responses develop rapidly, within 3 to 4 days, and are critical for preventing blood-borne pathogens from evolving into life-threatening infections. The interaction of BAFF, also known as BLyS, with its receptors BAFFR and TACI on B cells is critical for B cell homeostasis and function. Using a synthetic polysaccharide antigen, it has previously been shown that TACI is critical for T cell-independent antibody responses. To examine the role of BAFFR and TACI in T cell-independent antibody responses to an active infection, we utilized the Borrelia hermsii infection system. In this infection system, T cell-independent responses mediated by the B1b cell subset are critical for controlling bacteremia. We found that B1b cells express BAFFR and TACI and that the surface expression of both receptors is upregulated on B1b cells following exposure to whole B. hermsii cells. Surprisingly, we found that TACI(-/-) mice are not impaired either in specific antibody responses to B. hermsii or in controlling B. hermsii bacteremia. In contrast, TACI-deficient mice immunized with heat-killed type 3 serotype pneumococcus cells are impaired in generating pneumococcal polysaccharide-specific responses and succumb to challenge with live type 3 serotype pneumococcus, indicating that TACI is required for T cell-independent antibody responses to bacterial-associated polysaccharides. Although we have found that TACI is dispensable for controlling B. hermsii infection, mice deficient in BAFFR or BAFF exhibit impairment in B. hermsii-specific IgM responses and clearance of bacteremia. Collectively, these data indicate a disparity in the roles for TACI and BAFFR in primary T cell-independent antibody responses to bacterial pathogens.

Fine structure of neurally differentiated iPS cells generated from a multiple sclerosis (MS) patient: a case study.

We compared the characteristics of neural cells derived from induced pluripotent stem (iPS) cells from a patient with multiple sclerosis versus neurally differentiated control iPS cells of a healthy individual. The iPS cells were differentiated toward the oligodendrocyte lineage using a four-step protocol established for the differentiation of embryonic stem cells. The resulting cell population was immunostained on day 112 of differentiation for the presence of oligodendrocytes and analyzed by transmission electron microscopy (TEM). Both patient and control samples resembled a mixed population of neural cells rather than oligodendroglia of high purity, including neural stem cell-like cells and possibly oligodendrocytes demonstrable by TEM.

Causal role of immune cells in major depressive disorder and bipolar disorder: Mendelian randomization (MR) study.

BACKGROUND: Major depressive disorder (MDD) and bipolar disorder (BD) are prevalent psychiatric conditions linked to inflammatory processes. However, it is unclear whether associations of immune cells with these disorders are likely to be causal. METHODS: We used two-sample Mendelian randomization (MR) approach to investigate the relationship between 731 immune cells and the risk of MDD and BD. Rigorous sensitivity analyses are conducted to assess the reliability, heterogeneity, and horizontal pleiotropy of the findings. RESULTS: Genetically-predicted CD27 on IgD+ CD38- unswitched memory B cell (inverse variance weighting (IVW): odds ratio (OR) [95 %]: 1.017 [1.007 to 1.027], p = 0.001), CD27 on IgD+ CD24+ B cell (IVW: OR [95 %]: 1.021 [1.011 to 1.031], p = 4.821E-05) and other 12 immune cells were associated with increased risk of MDD in MR, while HLA DR++ monocyte %leukocyte (IVW: OR [95 %]: 0.973 [0.948 to 0.998], p = 0.038), CD4 on Central Memory CD4+ T cell (IVW: OR [95 %]: 0.979 [0.963 to 0.995], p = 0.011) and other 13 immune cells were associated with decreased risk of MDD in MR. Additionally, CD33+ HLA DR+ Absolute Count (IVW: OR [95 %]: 1.022[1.007 to 1.036], p = 0.007), CD28+ CD45RA- CD8+ T cell %T cell (IVW: OR [95 %]: 1.024 [1.008 to 1.041], p = 0.004) and other 18 immune cells were associated with increased risk of BD in MR, while CD62L on CD62L+ myeloid Dendritic Cell (IVW: OR [95 %]: 0.926 [0.871 to 0.985], p = 0.014), IgD- CD27- B cell %lymphocyte (IVW: OR [95 %]: 0.918 [0.880 to 0.956], p = 4.654E-05) and other 13 immune cells were associated with decreased risk of BD in MR. CONCLUSIONS: This MR study provides robust evidence supporting a causal relationship between immune cells and the susceptibility to MDD and BD, offering valuable insights for future clinical investigations. Experimental studies are also required to further examine causality, mechanisms, and treatment potential for these immune cells for MDD and BD.

The apparent loss of PRC2 chromatin occupancy as an artifact of RNA depletion.

RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favor and against this idea. RNase treatment of chromatin is commonly used to study RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. RNase A treatment during chromatin immunoprecipitation (ChIP) reduces chromatin occupancy of the H3K27me3 methyltransferase Polycomb repressive complex 2 (PRC2). This led to suggestions of an "RNA bridge" between PRC2 and chromatin. Here, we show that RNase A treatment during ChIP causes the apparent loss of all facultative heterochromatin, including both PRC2 and H3K27me3 genome-wide. We track this observation to a gain of DNA from non-targeted chromatin, sequenced at the expense of DNA from facultative heterochromatin, which reduces ChIP signals. Our results emphasize substantial limitations in using RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay.

Effects of Interleukin-19 overexpression in the medial prefrontal cortex on anxiety-related behaviors, BDNF expression and p38/JNK/ERK pathways.

Anxiety is a prevalent mental illness known for its high incidence, comorbidity, and tendency to recur, posing significant societal and individual burdens. Studies have highlighted Interleukin-19 (IL-19) as having potential relevance in neuropsychiatric disorders. Our previous research revealed that IL-19 overexpression in colonies exacerbated anxiety-related behaviors induced by dextran sodium sulfate/stress. However, the precise role and molecular mechanisms of IL-19 in anxiety regulation remain uncertain. In this study, we initiated an acute restraint stress (ARS)-induced anxious mouse model and identified heightened expression of IL-19 and IL-20Rα in the medial prefrontal cortex (mPFC) of ARS mice. Notably, IL-19 and IL-20Rα were predominantly present in the excitatory pyramidal neurons of the mPFC under both basal and ARS conditions. Utilizing the adeno-associated virus (AAV) strategy, we demonstrated that IL-19 overexpression in the mPFC induced anxiety-related behaviors and elevated stress susceptibility. Additionally, we observed decreased protein levels of brain-derived neurotrophic factor (BDNF) and postsynaptic density protein 95 (PSD95) in the mPFC of IL-19 overexpression mice, accompanied by reduced phosphorylation of in the p38, JNK, and Erk signaling pathways. These findings emphasize the role of IL-19 in modulating anxiety-related behaviors within the mPFC and suggest its potential as a pathological gene and therapeutic target for anxiety.

The renoprotective efficacy and safety of genetically-engineered human bone marrow-derived mesenchymal stromal cells expressing anti-fibrotic cargo.

BACKGROUND: Kidney fibrosis is a hallmark of chronic kidney disease (CKD) and compromises the viability of transplanted human bone marrow-derived mesenchymal stromal cells (BM-MSCs). Hence, BM-MSCs were genetically-engineered to express the anti-fibrotic and renoprotective hormone, human relaxin-2 (RLX) and green fluorescent protein (BM-MSCs-eRLX + GFP), which enabled BM-MSCs-eRLX + GFP delivery via a single intravenous injection. METHODS: BM-MSCs were lentiviral-transduced with human relaxin-2 cDNA and GFP, under a eukaryotic translation elongation factor-1α promoter (BM-MSCs-eRLX + GFP) or GFP alone (BM-MSCs-eGFP). The ability of BM-MSCs-eRLX + GFP to differentiate, proliferate, migrate, produce RLX and cytokines was evaluated in vitro, whilst BM-MSC-eRLX + GFP vs BM-MSCs-eGFP homing to the injured kidney and renoprotective effects were evaluated in preclinical models of ischemia reperfusion injury (IRI) and high salt (HS)-induced hypertensive CKD in vivo. The long-term safety of BM-MSCs-RLX + GFP was also determined 9-months after treatment cessation in vivo. RESULTS: When cultured for 3- or 7-days in vitro, 1 × 106 BM-MSCs-eRLX + GFP produced therapeutic RLX levels, and secreted an enhanced but finely-tuned cytokine profile without compromising their proliferation or differentiation capacity compared to naïve BM-MSCs. BM-MSCs-eRLX + GFP were identified in the kidney 2-weeks post-administration and retained the therapeutic effects of RLX in vivo. 1-2 × 106 BM-MSCs-eRLX + GFP attenuated the IRI- or therapeutically abrogated the HS-induced tubular epithelial damage and interstitial fibrosis, and significantly reduced the HS-induced hypertension, glomerulosclerosis and proteinuria. This was to an equivalent extent as RLX and BM-MSCs administered separately but to a broader extent than BM-MSCs-eGFP or the angiotensin-converting enzyme inhibitor, perindopril. Additionally, these renoprotective effects of BM-MSCs-eRLX + GFP were maintained in the presence of perindopril co-treatment, highlighting their suitability as adjunct therapies to ACE inhibition. Importantly, no major long-term adverse effects of BM-MSCs-eRLX + GFP were observed. CONCLUSIONS: BM-MSCs-eRLX + GFP produced greater renoprotective and therapeutic efficacy over that of BM-MSCs-eGFP or ACE inhibition, and may represent a novel and safe treatment option for acute kidney injury and hypertensive CKD.

Human adipose-derived mesenchymal stem cells engineered to secrete IL-10 inhibit APC function and limit CNS autoimmunity.

Interleukin (IL)-10 is an important immunoregulatory cytokine shown to impact inflammatory processes as manifested in patients with multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). Several lines of evidence indicate that the effectiveness of IL-10-based therapies may be dependent on the timing and mode of delivery. In the present study we engineered the expression of IL-10 in human adipose-derived mesenchymal stem cells (Adi-IL-10-MSCs) and transplanted these cells early in the disease course to mice with EAE. Adi-IL-10-MSCs transplanted via the intraperitoneal route prevented or delayed the development of EAE. This protective effect was associated with several anti-inflammatory response mechanisms, including a reduction in peripheral T-cell proliferative responses, a decrease in pro-inflammatory cytokine secretion as well as a preferential inhibition of Th17-mediated neuroinflammation. In vitro analyses revealed that Adi-IL-10-MSCs inhibited the phenotypic maturation, cytokine production and antigen presenting capacity of bone marrow-derived myeloid dendritic cells, suggesting that the mechanism of action may involve an indirect effect on pathogenic T-cells via the modulation of antigen presenting cell function. Collectively, these results suggest that early intervention with gene modified Adi-MSCs may be beneficial for the treatment of autoimmune diseases such as MS.

19-[(1'S,4'R)-4'-Hydroxy-1'-methoxy-2'-oxopentyl]geldanamycin, a natural geldanamycin analogue from Streptomyces hygroscopicus 17997.

A novel natural geldanamycin analogue was discovered in Streptomyces hygroscopicus 17997. Its 4,5-dihydro form was also identified in the gdmP gene disruption mutant of Streptomyces hygroscopicus 17997. The structures of the two compounds were determined to be 19-[(1'S,4'R)-4'-hydroxy-1'-methoxy-2'-oxopentyl]geldanamycin (1) and 19-[(1'S,4'R)-4'-hydroxy-1'-methoxy-2'-oxopentyl]-4,5-dihydrogeldanamycin (2), respectively, by extensive spectroscopic data analysis, including 2D NMR, modified Mosher's method, and electronic circular dichroism. Compared to geldanamycin, 1 and 2 showed increased water solubility and decreased cytotoxicity against HepG2 cells.