Efficient CRISPR-Cas9 mediated gene disruption in primary erythroid progenitor cells

The study of isolated primary progenitor cells offers great insight into developmental biology and human disease. In particular, ex vivo culture of isolated primary erythroid progenitor cells replicates the differentiation events that occur during in vivo erythropoiesis. Herein we report a high-efficiency method for CRISPR-Cas9 mediated gene disruption in isolated primary erythroid progenitor cells. We use this method to generate the novel result that Lmna is required in terminal erythroid differentiation.Frederick Lovejoy (Research Grant)National Institutes of Health (U.S.) (grant NIH/NHLBI 2 P01 HL032262-25

Noninvasive immuno-PET imaging of CD8 + T cell behavior in influenza A virus-infected mice

Immuno-positron emission tomography (immuno-PET) is a noninvasive imaging method that enables tracking of immune cells in living animals. We used a nanobody that recognizes mouse CD8α and labeled it wit

A class II MHC-targeted vaccine elicits immunity against SARS-CoV-2 and its variants

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (Spik

Enhanced phosphocholine metabolism is essential for terminal erythropoiesis

Red cells contain a unique constellation of membrane lipids. Although much is known about regulated protein expression, the regulation of lipid metabolism during erythropoiesis is poorly studied. Here, we show that transcription of PHOSPHO1, a phosphoethanolamine and phosphocholine phosphatase that mediates the hydrolysis of phosphocholine to choline, is strongly upregulated during the terminal stages of erythropoiesis of both human and mouse erythropoiesis, concomitant with increased catabolism of phosphatidylcholine (PC) and phosphocholine as shown by global lipidomic analyses of mouse and human terminal erythropoiesis. Depletion of PHOSPHO1 impaired differentiation of fetal mouse and human erythroblasts, and, in adult mice, depletion impaired phenylhydrazine-induced stress erythropoiesis. Loss of PHOSPHO1 also impaired phosphocholine catabolism in mouse fetal liver progenitors and resulted in accumulation of several lipids; adenosine triphosphate (ATP) production was reduced as a result of decreased oxidative phosphorylation. Glycolysis replaced oxidative phosphorylation in PHOSPHO1-knockout erythroblasts and the increased glycolysis was used for the production of serine or glycine. Our study elucidates the dynamic changes in lipid metabolism during terminal erythropoiesis and reveals the key roles of PC and phosphocholine metabolism in energy balance and amino acid supply.United States. Defense Advanced Research Projects Agency (Contract HR0011-14-2-0005)National Heart, Lung, and Blood Institute (Grant 2 P01 HL032262-25

Transcriptional divergence and conservation of human and mouse erythropoiesis

Thesis: S.M., Massachusetts Institute of Technology, Department of Biological Engineering, 2014.Cataloged from PDF version of thesis.Includes bibliographical references.Mouse models have been used extensively for decades and have been instrumental in improving our understanding of mammalian erythropoiesis. Nonetheless, there are several examples of variation between human and mouse erythropoiesis. We performed a comparative global gene expression study using data from morphologically identical stage-matched sorted populations of human and mouse erythroid precursors from early to late erythroblasts. Induction and repression of major transcriptional regulators of erythropoiesis, as well as major erythroid-important proteins, are largely conserved between the species. In contrast, at a global level we identified a significant extent of divergence between the species, both at comparable stages and in the transitions between stages, especially for the 500 most highly expressed genes during development. This suggests that the response of multiple developmentally regulated genes to key erythroid transcriptional regulators represents an important modification that has occurred in the course of erythroid evolution. In developing a systematic framework to understand and study conservation and divergence between human and mouse erythropoiesis, we show how mouse models can fail to mimic specific human diseases and provide predictions for translating findings from mouse models to potential therapies for human disease.by Novalia Pishesha.S.M

Engineered red blood cells and their applications

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018Cataloged from PDF version of thesis.Includes bibliographical references.The humble red blood cell (RBC) is the most abundant cell in the human body. Every second, a normal adult generates some 2.5 million RBCs, which subsequently circulate through the blood vessels for a lifespan of 50 or 120 days in mouse and human, respectively. RBCs are also unique in that they are completely enucleated once fully mature. These two characteristics exist as distinct assets for cellular therapy applications utilizing RBCs as a platform, enabling long-lasting availability in vivo and the ability to genetically modify precursor cells without worry of the terminally differentiated progeny carrying any foreign genetic material. The first part of this thesis is devoted to the establishment of methodologies that allow for the covalent attachment of both natural and synthetic cargoes to the surface of red blood cells without compromising its biological properties. This system employs genetic engineering and sortase A, a bacterial transpeptidases.We show that this strategy is able to efficiently engineer both mature mouse and human RBCs in a site-specific and covalent manner. The next portion of this work describes how these established methodologies can be mixed and matched according to the diverse needs of engineered RBC applications. We provide a proof of concept that utilizes engineered RBCs to prolong prophylactic protection against deadly toxins. By expressing chimeric proteins of single domain antibodies (VHHs) against botulinum neurotoxin A (BoNT/A) with RBC-specific proteins, we demonstrated that mouse RBCs expressing anti-BoNT/A VHHs can provide resistance up to 10,000 times the lethal dose (LD₅₀) of BoNT/A. We validate this finding by repeating our results in a human RBC culture system that we have improved to achieve 90% enucleation, illustrating the broad translatability of our strategy for therapeutic applications.Finally, drawing upon knowledge that the body clears 2.5 millions RBCs every second to maintain homeostasis, we use sortase to attach disease-associated autoantigens to genetically engineered and to unmodified red blood cells (RBCs). Such modified RBCs masquerade with these autoantigens as their own, and hijack the non-inflammatory nature of the RBC clearance pathway to promote tolerance to their carried payload. We show that this blunts the immune contribution of major subsets of immune effector cells (B cells, CD4+ and CD8+ T cells) in an antigen-specific manner. Transfusion of RBCs expressing self-antigen epitopes alleviates and even prevents signs of disease in an experimental system for autoimmune encephalomyelitis, and also maintains normoglycemia in a mouse model of type 1 diabetes, highlighting the potential of engineered RBCs for treating autoimmune diseases.Taken together, the results of applying our engineered RBCs in areas of both acute infectious and toxic agents, as well as for longer-term chronic and autoimmune diseases, hint at the tremendous potential of this system, and we have only begun to scratch the surface.by Novalia Pishesha.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Biological Engineerin

Age dependent increase in the levels of osteopontin inhibits skeletal muscle regeneration.

Skeletal muscle regeneration following injury is accompanied by rapid infiltration of macrophages, which play a positive role in muscle repair. Increased chronic inflammation inhibits the regeneration of dystrophic muscle, but the properties of inflammatory cells are not well understood in the context of normal muscle aging. This work uncovers pronounced age-specific changes in the expression of osteopontin (OPN) in CD11b+ macrophages present in the injured old muscle as well as in the blood serum of old injured mice and in the basement membrane surrounding old injured muscle fibers. Furthermore, young CD11b+ macrophages enhance regenerative capacity of old muscle stem cells even when old myofibers and old sera are present; and neutralization of OPN similarly rejuvenates the myogenic responses of old satellite cells in vitro and notably, in vivo. This study highlights potential mechanisms by which age related inflammatory responses become counter-productive for muscle regeneration and suggests new strategies for enhancing muscle repair in the old

Nanobodies as in vivo, non-invasive, imaging agents

In vivo imaging has become in recent years an incredible tool to study biological events and has found critical applications in diagnostic medicine. Although a lot of efforts and applications have been achieved using monoclonal antibodies, other types of delivery agents are being developed. Among them, VHHs, antigen binding fragments derived from camelid heavy chain–only antibodies, also known as nanobodies, have particularly attracted attention. Indeed, their stability, fast clearance, good tissue penetration, high solubility, simple cloning and recombinant production make them attractive targeting agents for imaging modalities such as PET, SPECT or Infra-Red. In this review, we discuss the pioneering work that has been carried out using VHHs and summarize the recent developments that have been made using nanobodies for in vivo, non-invasive, imaging

Material Viscoelastic Properties Modulate the Mesenchymal Stem Cell Secretome for Applications in Hematopoietic Recovery

© 2017 American Chemical Society. Human mesenchymal stem cells (MSCs) exhibit morphological and phenotypic changes that correlate with mechanical cues presented by the substratum material to which those cells adhere. Such mechanosensitivity has been explored in vitro to promote differentiation of MSCs along tissue cell lineages for direct tissue repair. However, MSCs are increasingly understood to facilitate indirect tissue repair in vivo through paracrine signaling via secreted biomolecules. Here we leveraged cell-material interactions in vitro to induce human bone marrow-derived MSCs to preferentially secrete factors that are beneficial to hematopoietic cell proliferation. Specifically, we varied the viscoelastic properties of cell-culture-compatible polydimethylsiloxane (PDMS) substrata to demonstrate modulated MSC expression of biomolecules, including osteopontin, a secreted phosphoprotein implicated in tissue repair and regeneration. We observed an approximately 3-fold increase in expression of osteopontin for MSCs on PDMS substrata of lowest stiffness (elastic moduli 1). A specific subpopulation of these cells, shown previously to express increased osteopontin in vitro and to promote bone marrow recovery in vivo, also exhibited up to a 5-fold increase in osteopontin expression when grown on compliant PDMS relative to heterogeneous MSCs on polystyrene. Importantly, this mechanically modulated increase in protein expression preceded detectable changes in the terminal differentiation capacity of MSCs. In coculture with human CD34+ hematopoietic stem and progenitor cells (HSPCs) that repopulate the blood cell lineages, these mechanically modulated MSCs promoted in vitro proliferation of HSPCs without altering the multipotency for either myeloid or lymphoid lineages. Cytokine and protein expression by human MSCs can thus be manipulated directly by mechanical cues conferred by the material substrata prior to and instead of tissue lineage differentiation. This approach enables enhanced in vitro production of both mesenchymal and hematopoietic stem and progenitor cells that aid regenerative clinical applications

Transcriptional divergence and conservation of human and mouse erythropoiesis

Mouse models have been used extensively for decades and have been instrumental in improving our understanding of mammalian erythropoiesis. Nonetheless, there are several examples of variation between human and mouse erythropoiesis. We performed a comparative global gene expression study using data from morphologically identical stage-matched sorted populations of human and mouse erythroid precursors from early to late erythroblasts. Induction and repression of major transcriptional regulators of erythropoiesis, as well as major erythroid-important proteins, are largely conserved between the species. In contrast, at a global level we identified a significant extent of divergence between the species, both at comparable stages and in the transitions between stages, especially for the 500 most highly expressed genes during development. This suggests that the response of multiple developmentally regulated genes to key erythroid transcriptional regulators represents an important modification that has occurred in the course of erythroid evolution. In developing a systematic framework to understand and study conservation and divergence between human and mouse erythropoiesis, we show how mouse models can fail to mimic specific human diseases and provide predictions for translating findings from mouse models to potential therapies for human disease.National Institutes of Health (U.S.) (Grant P01 HL32262)American Association of University Women (Fellowship)Philanthropic Educational Organization (International Peace Scholarship Fund)Schlumberger Foundation. Faculty for the Futur