Increases in the number of cell therapies in the preclinical and clinical phases have prompted the need for reliable and non-invasive assays to validate transplant function in clinical biomanufacturing. We developed a robust characterization methodology composed of quantitative bright-field absorbance microscopy (QBAM) and deep neural networks (DNNs) to non-invasively predict tissue function and cellular donor identity. The methodology was validated using clinical-grade induced pluripotent stem cell derived retinal pigment epithelial cells (iPSC-RPE). QBAM images of iPSC-RPE were used to train DNNs that predicted iPSC-RPE monolayer transepithelial resistance, predicted polarized vascular endothelial growth factor (VEGF) secretion, and matched iPSC-RPE monolayers to the stem cell donors. DNN predictions were supplemented with traditional machine learning algorithms that identified shape and texture features of single cells that were used to predict tissue function and iPSC donor identity. These results demonstrate non-invasive cell therapy characterization can be achieved with QBAM and machine learning.
Nicholas J. Schaub, Nathan A. Hotaling, Petre Manescu, Sarala Padi, Qin Wan, Ruchi Sharma, Aman George, Joe Chalfoun, Mylene Simon, Mohamed Ouladi, Carl G. Simon, Jr., Peter Bajcsy, Kapil Bharti
Parkinson's disease (PD) is a neurodegenerative disorder associated with loss of striatal dopamine, secondary to degeneration of midbrain dopamine (mDA) neurons in the substantia nigra, rendering cell transplantation a promising therapeutic strategy. To establish human induced pluripotent stem cell (hiPSC)-based autologous cell therapy, we report a platform of core techniques for the production of mDA progenitors as a safe and effective therapeutic product. First, by combining metabolism-regulating microRNAs with reprogramming factors, we developed a method to more efficiently generate clinical grade iPSCs, as evidenced by genomic integrity and unbiased pluripotent potential. Second, we established a “spotting”-based in vitro differentiation methodology to generate functional and healthy mDA cells in a scalable manner. Third, we developed a chemical method that safely eliminates undifferentiated cells from the final product. Dopaminergic cells thus produced express high levels of characteristic mDA markers, produce and secrete dopamine, and exhibit electrophysiological features typical of mDA cells. Transplantation of these cells into rodent models of PD robustly restores motor dysfunction and reinnervates host brain, while showing no evidence of tumor formation or redistribution of the implanted cells. We propose that this platform is suitable for the successful implementation of human personalized autologous cell therapy for PD.
Bin Song, Young Cha, Sanghyeok Ko, Jeha Jeon, Nayeon Lee, Hyemyung Seo, Kyung-joon Park, In-Hee Lee, Claudia Lopes, Melissa Feitosa, María José Luna, Jin Hyuk Jung, Jisun Kim, Dabin Hwang, Bruce Cohen, Martin Teicher, Pierre Leblanc, Bob Carter, Jeffrey H. Kordower, Vadim Y. Bolshakov, Sek Won Kong, Jeffrey S. Schweitzer, Kwang-Soo Kim
Interventions to prevent HIV-1 infection and alternative tools in HIV cure therapy remain pressing goals. Recently, numerous broadly neutralizing HIV-1 monoclonal antibodies (bNAbs) have been developed which possess the characteristics necessary for potential prophylactic or therapeutic approaches. However, formulation complexities especially for multi-antibody deliveries, long infusion times, and production issues could limit the use of these bNAbs when deployed globally impacting their potential application. Here, we describe an approach utilizing synthetic DNA-encoded monoclonal antibodies (dMAbs) for direct in vivo production of prespecified neutralizing activity. We designed 16 different bNAbs as dMAbs cassettes and studied their activity in small and large animals. Sera from animals administered dMAbs neutralized multiple HIV-1 isolates with similar activity to their parental recombinant MAbs. Delivery of multiple dMAbs to a single animal led to increased neutralization breadth. Two dMAbs, PGDM1400 and PGT121, were advanced into non-human primates for study. High peak circulating levels (between 6-34µg/ml) of these dMAbs were measured and the sera of all animals displayed broad neutralizing activity. The dMAb approach provides an important local delivery platform for the in vivo generation of HIV-1 bNAbs and for other infectious disease antibodies.
Megan C. Wise, Ziyang Xu, Edgar Tello-Ruiz, Charles Beck, Aspen Trautz, Ami Patel, Sarah T.C. Elliott, Neethu Chokkalingam, Sophie Kim, Melissa G. Kerkau, Kar Muthumani, Jingjing Jiang, Paul Fisher, Stephany J. Ramos, Trevor R.F. Smith, Janess Mendoza, Kate E. Broderick, David C. Montefiori, Guido Ferrari, Daniel W. Kulp, Laurent Humeau, David B. Weiner
Successful infection by mucosal pathogens requires overcoming the mucus barrier. To better understand this key step, we performed a survey of the interactions between human respiratory mucus and the human pathogen S. pneumoniae. Pneumococcal adherence to adult human nasal fluid was seen only by isolates expressing pilus-1. Robust binding was independent of pilus-1 adhesive properties but required Fab-dependent recognition of RrgB, the pilus shaft protein, by naturally-acquired secretory immunoglobulin A (sIgA). Pilus-1 binding by specific sIgA led to bacterial agglutination, but adherence required interaction of agglutinated pneumococci and entrapment in mucus particles. To test the effect of these interactions in vivo, pneumococci were preincubated with human sIgA prior to intranasal challenge in a mouse model of colonization. sIgA-treatment resulted in rapid immune exclusion of pilus-expressing pneumococci. Our findings predict that immune exclusion would select for non-piliated isolates in individuals who acquired RrgB-specific sIgA from prior episodes of colonization with piliated strains. Accordingly, genomic data comparing isolates carried by mothers and their children showed that mothers are less likely to be colonized with pilus-expressing strains. Our study provides a specific example of immune exclusion involving naturally-acquired antibody in the human host, a major factor driving pneumococcal adaptation.
Ulrike Binsker, John A. Lees, Alexandria J. Hammond, Jeffrey N. Weiser
Glucocorticoids (GCs) are a central component of therapy for patients with T-cell acute lymphoblastic leukemia (T-ALL) and while resistance to GCs is a strong negative prognostic indicator in T-ALL, mechanisms of GC resistance remain poorly understood. Using diagnostic samples from patients enrolled on the frontline Children’s Oncology Group (COG) T-ALL clinical trial AALL1231, we demonstrated that one-third of primary T-ALLs were resistant to GCs when cultured in the presence of interleukin-7 (IL7), a cytokine that is critical for normal T-cell function and that plays a well-established role in leukemogenesis. We demonstrated that in these T-ALLs and in distinct populations of normal developing thymocytes, GCs paradoxically induced their own resistance by promoting upregulation of IL7 receptor (IL7R) expression. In the presence of IL7, this augmented downstream signal transduction resulting in increased STAT5 transcriptional output and upregulation of the pro-survival protein BCL-2. Taken together, we demonstrated that IL7 mediates an intrinsic and physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALLs and is reversible with targeted inhibition of the IL7R/JAK/STAT5/BCL-2 axis.
Lauren K. Meyer, Benjamin J. Huang, Cristina Delgado-Martin, Ritu P. Roy, Aaron Hechmer, Anica M. Wandler, Tiffaney L. Vincent, Paolo Fortina, Adam B. Olshen, Brent L. Wood, Terzah M. Horton, Kevin M. Shannon, David T. Teachey, Michelle L. Hermiston
X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia (XMEN) disease is caused by deficiency of the magnesium transporter 1 gene (MAGT1). We studied 23 XMEN patients, 8 of whom were EBV-naïve. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum, and increased CD4-CD8-B220-TCRalpha/beta+ T (abDNT) cells, in addition to the previously described features of an inverted CD4:CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the “Natural-Killer Group 2, member D” (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We investigated XMEN patients and autoimmune lymphoproliferative syndrome (ALPS) patients by deep immunophenotyping (32 immune markers) using Time of Flight Mass Cytometry (CyTOF). Our analysis revealed that the abundance of two populations of naïve B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and normal individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.
Juan C. Ravell, Mami Matsuda-Lennikov, Samuel D. Chauvin, Juan Zou, Matthew Biancalana, Sally J. Deeb, Susan Price, Helen C. Su, Giulia Notarangelo, Ping Jiang, Aaron Morawski, Chrysi Kanellopoulou, Kyle W. Binder, Ratnadeep Mukherjee, James T. Anibal, Brian Sellers, Lixin Zheng, Tingyan He, Alex B. George, Stefania Pittaluga, Astin Powers, David E. Kleiner, Devika Kapuria, Marc Ghany, Sally Hunsberger, Jeffrey I. Cohen, Gulbu Uzel, Jenna Bergerson, Lynne Wolfe, Camilo Toro, William Gahl, Les R. Folio, Helen Matthews, Pam Angelus, Ivan K. Chinn, Jordan S. Orange, Claudia M. Trujillo-Vargas, Jose Luis Franco, Julio Orrego-Arango, Sebastian Gutiérrez-Hincapié, Niraj Chandrakant Patel, Kimiyo Raymond, Turkan Patiroglu, Ekrem Unal, Musa Karakukcu, Alexandre G.R. Day, Pankaj Mehta, Evan Masutani, Suk S. De Ravin, Harry L. Malech, Grégoire Altan-Bonnet, V. Koneti Rao, Matthias Mann, Michael J. Lenardo
Leptomeningeal anastomoses or pial collateral vessels play a critical role in cerebral blood flow (CBF) restoration following ischemic stroke. The magnitude of this adaptive response is postulated to be controlled by the endothelium, although the underlying molecular mechanisms remain under investigation. Here we demonstrated that endothelial genetic deletion, using EphA4f/f/Tie2-Cre and EphA4f/f/VeCahderin-CreERT2 mice and vessel painting strategies, implicated EphA4 receptor tyrosine kinase as a major suppressor of pial collateral remodeling, CBF and functional recovery following permanent middle cerebral artery occlusion. Pial collateral remodeling is limited by the cross talk between EphA4-Tie2 signaling in vascular endothelial cells, which is mediated through p-Akt regulation. Furthermore, peptide inhibition of EphA4 resulted in acceleration of the pial arteriogenic response. Our findings demonstrate EphA4 is a negative regulator of Tie2 receptor signaling which limits pial collateral arteriogenesis following cerebrovascular occlusion. Therapeutic targeting of EphA4 and/or Tie2 represents an attractive new strategy for improving collateral function, neural tissue health and functional recovery following ischemic stroke.
Benjamin Okyere, William A. Mills III, Xia Wang, Michael Chen, Jiang Chen, Amanda Hazy, Yun Qian, John B. Matson, Michelle H. Theus
Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1-ENG-Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K-Akt-mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine-kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs—including in HHT patient blood outgrowth ECs—and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in HHT patients.
Santiago Ruiz, Haitian Zhao, Pallavi Chandakkar, Julien Papoin, Hyunwoo Choi, Aya Nomura-Kitabayashi, Radhika Patel, Matthew Gillen, Li Diao, Prodyot K. Chatterjee, Mingzhu He, Yousef Al-Abed, Ping Wang, Christine N. Metz, S. Paul Oh, Lionel Blanc, Fabien Campagne, Philippe Marambaud
EGFR mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFRT790M and EGFRC797S. It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (pSTAT3) in EGFRT790M and EGFRC797S tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a pSTAT3-dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor resistant tumors, with EGFRT790M and EGFRC797S mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib and osimertinib respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib treated patients after disease progression.
Emilie Bousquet Mur, Sara Bernardo, Laura Papon, Maicol Mancini, Eric Fabbrizio, Marion Goussard, Irene Ferrer, Anais Giry, Xavier Quantin, Jean-Louis Pujol, Olivier Calvayrac, Herwig P. Moll, Yaël Glasson, Nelly Pirot, Andrei Turtoi, Marta Cañamero, Kwok-Kin Wong, Yosef Yarden, Emilio Casanova, Jean-Charles Soria, Jacques Colinge, Christian W. Siebel, Julien Mazieres, Gilles Favre, Luis Paz-Ares, Antonio Maraver
The corneocyte lipid envelope composed of covalently bound ceramides and fatty acids is important to the integrity of the permeability barrier in the stratum corneum, and its absence is a prime structural defect in various skin diseases associated with defective skin barrier function. SDR9C7 encodes a short chain dehydrogenase/reductase family 9C member 7 (SDR9C7) recently found mutated in ichthyosis. In a patient with SDR9C7 mutation and a mouse Sdr9c7 knockout model we show loss of covalent binding of epidermal ceramides to protein, a structural fault in the barrier. For reasons unresolved, protein binding requires lipoxygenase-catalyzed transformations of linoleic acid (18:2) esterified in ω-O-acylceramides. In Sdr9c7-/- epidermis, quantitative LC-MS assays revealed almost complete loss of a species of ω-O-acylceramide esterified with linoleate-9,10-trans-epoxy-11E-13-ketone; other acylceramides related to the lipoxygenase pathway were in higher abundance. Recombinant SDR9C7 catalyzed NAD+-dependent dehydrogenation of linoleate 9,10-trans-epoxy-11E-13-alcohol to the corresponding 13-ketone, while ichthyosis mutants were inactive. We propose, therefore, that the critical requirement for lipoxygenases and SDR9C7 is in producing acylceramide containing the 9,10-epoxy-11E-13-ketone, a reactive moiety known for its non-enzymatic coupling to protein. This suggests a mechanism for coupling of ceramide to protein and provides important insights into skin barrier formation and pathogenesis.
Takuya Takeichi, Tetsuya Hirabayashi, Yuki Miyasaka, Akane Kawamoto, Yusuke Okuno, Shijima Taguchi, Kana Tanahashi, Chiaki Murase, Hiroyuki Takama, Kosei Tanaka, William E. Boeglin, M. Wade Calcutt, Daisuke Watanabe, Michihiro Kono, Yoshinao Muro, Junko Ishikawa, Tamio Ohno, Alan R. Brash, Masashi Akiyama
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