Investigation of the impact of clonal hematopoiesis on severity and pathophysiology of COVID-19 in rhesus macaques.

Clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe respiratory failure with profound inflammation. Although risk factors for severe illness have been identified, definitive determinants remain elusive. Clonal hematopoiesis (CH), the expansion of hematopoietic stem and progenitor cells bearing acquired somatic mutations, is associated with advanced age and hyperinflammation. Given the similar age range and hyperinflammatory phenotype between frequent CH and severe COVID-19, CH could impact the risk of severe COVID-19. Human cohort studies have attempted to prove this relationship, but conclusions are conflicting. Rhesus macaques (RMs) are being utilized to test vaccines and therapeutics for COVID-19. However, RMs, even other species, have not yet been reported to develop late inflammatory COVID-19 disease. Here, RMs with either spontaneous DNMT3A or engineered TET2 CH along with similarly transplanted and conditioned controls were infected with SARS-CoV-2 and monitored until 12 days post-inoculation (dpi). Although no significant differences in clinical symptoms and blood counts were noted, an aged animal with natural DNMT3A CH died on 10 dpi. CH macaques showed evidence of sustained local inflammatory responses compared to controls. Interestingly, viral loads in respiratory tracts were higher at every timepoint in the CH group. Lung sections from euthanasia showed evidence of mild inflammation in all animals, while viral antigen was more frequently detected in the lung tissues of CH macaques even at the time of autopsy. Despite the lack of striking inflammation and serious illness, our findings suggest potential pathophysiological differences in RMs with or without CH upon SARS-CoV-2 infection.

Investigation of the Impact of Clonal Hematopoiesis on Severity and Pathophysiology of COVID-19 in Rhesus Macaques.

Clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe respiratory failure with profound inflammation. Although risk factors for severe illness have been identified, definitive determinants remain elusive. Clonal hematopoiesis (CH), the expansion of hematopoietic stem and progenitor cells bearing acquired somatic mutations, is associated with advanced age and hyperinflammation. Given the similar age range and hyperinflammatory phenotype between frequent CH and severe COVID-19, CH could impact the risk of severe COVID-19. Human cohort studies have attempted to prove this relationship, but conclusions are conflicting. Rhesus macaques (RMs) are being utilized to test vaccines and therapeutics for COVID-19. However, RMs, even other species, have not yet been reported to develop late inflammatory COVID-19 disease. Here, RMs with either spontaneous DNMT3A or engineered TET2 CH along with similarly transplanted and conditioned controls were infected with SARS-CoV-2 and monitored until 12 days post-inoculation (dpi). Although no significant differences in clinical symptoms and blood counts were noted, an aged animal with natural DNMT3A CH died on 10 dpi. CH macaques showed evidence of sustained local inflammatory responses compared to controls. Interestingly, viral loads in respiratory tracts were higher at every timepoint in the CH group. Lung sections from euthanasia showed evidence of mild inflammation in all animals, while viral antigen was more frequently detected in the lung tissues of CH macaques even at the time of autopsy. Despite the lack of striking inflammation and serious illness, our findings suggest potential pathophysiological differences in RMs with or without CH upon SARS-CoV-2 infection. Highlights: No evidence of association between CH and COVID-19 clinical severity in macaques.The presence of CH is associated with prolonged local inflammatory responses in COVID-19.SARS-CoV-2 persists longer in respiratory tracts of macaques with CH following infection.

A RUNX1-FPDMM rhesus macaque model reproduces the human phenotype and predicts challenges to curative gene therapies.

Germ line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous-mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.

Mesenchymal stem cells target microglia via galectin-1 production to rescue aged mice from olfactory dysfunction.

Olfactory loss has been considered as the earliest complication for the aging process while underlying mechanisms and therapeutic strategies remain unclear. Given the correlation between microglial activation and olfactory dysfunction, here we investigated whether the immunomodulatory action of mesenchymal stem cells (MSCs) can rescue the olfactory impairment in old mice. The intranasal delivery of MSCs limited microglial activation and neuronal apoptosis in the olfactory bulb (OB), leading to improvement in olfaction. MSCs down-regulated the proportion of CD86+ microglia and prevented the maturation of cathepsin S, one of the inflammatory mediators in olfactory impairment, via the suppression of p38 MAPK signaling. Notably, old astrocytes could not prevent excessive microgliosis because the endogenous production of Galectin-1 (Gal1), one of the key microglia regulators secreted by astrocytes, was not sufficiently upregulated in the aged brain despite the presence of reactive astrogliosis. Considering that Gal1 is known as a potent paracrine factor of MSCs, we investigated whether MSC-derived Gal1 could compensate for defective astrocyte function in terms of microglial regulation. MSCs and their culture supernatant (MSC-CM) could regulate the direction of microglial differentiation by impeding the polarization towards the pro-inflammatory M1 type; notably, a selective Gal1 inhibitor OTX008 could hinder this phenomenon, indicating that Gal1 is involved in immunomodulation exerted by MSCs. Also, acute microglial activation within the OB upon LPS infusion was attenuated by MSC-CM in a Gal1-dependent manner. Our study demonstrates the therapeutic benefit of MSCs on age-related olfactory dysfunction and suggests Gal1 as a key mediator of the anti-inflammatory action of MSCs.

A macaque clonal hematopoiesis model demonstrates expansion of TET2-disrupted clones and utility for testing interventions.

Individuals with age-related clonal hematopoiesis (CH) are at greater risk for hematologic malignancies and cardiovascular diseases. However, predictive preclinical animal models to recapitulate the spectrum of human CH are lacking. Through error-corrected sequencing of 56 human CH/myeloid malignancy genes, we identified natural CH driver mutations in aged rhesus macaques matching genes somatically mutated in human CH, with DNMT3A mutations being the most frequent. A CH model in young adult macaques was generated via autologous transplantation of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene-edited hematopoietic stem and progenitor cells (HSPCs), targeting the top human CH genes with loss-of-function (LOF) mutations. Long-term follow-up revealed reproducible and significant expansion of multiple HSPC clones with heterozygous TET2 LOF mutations, compared with minimal expansion of clones bearing other mutations. Although the blood counts of these CH macaques were normal, their bone marrows were hypercellular and myeloid-predominant. TET2-disrupted myeloid colony-forming units isolated from these animals showed a distinct hyperinflammatory gene expression profile compared with wild type. In addition, mature macrophages purified from the CH macaques showed elevated NLRP3 inflammasome activity and increased interleukin-1ß (IL-1ß) and IL-6 production. The model was used to test the impact of IL-6 blockage by tocilizumab, documenting a slowing of TET2-mutated expansion, suggesting that interruption of the IL-6 axis may remove the selective advantage of mutant HSPCs. These findings provide a model for examining the pathophysiology of CH and give insights into potential therapeutic interventions.

Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.

The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-seq and ISP for a CD33 guide RNA (gRNA) with thousands of off-target sites predicted by ISP and CIRCLE-seq. We found poor correlation between the sites predicted by the two methods. When almost 500 sites predicted by each method were analyzed by error-corrected sequencing of hematopoietic cells following transplantation, 19 off-target sites revealed insertion or deletion mutations. Of these sites, 8 were predicted by both methods, 8 by CIRCLE-seq only, and 3 by ISP only. The levels of cells with these off-target edits exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In addition, we utilized an unbiased method termed CAST-seq to search for translocations between the on-target site and off-target sites present in animals following transplantation, detecting one specific translocation that persisted in blood cells for at least 1 year following transplantation. In conclusion, neither CIRCLE-seq or ISP predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods, may be required for optimizing safety of clinical development.

Respiratory and Systemic Toxicity of Inhaled Artificial Asian Sand Dust in Pigs.

Air pollution, particularly caused by Asian sand dust (ASD) and particulate matter (PM), has become one of the leading threats to public health. However, the majority of studies have primarily focused on epidemiological assessment, and in vivo toxicities of certain air pollutants have been poorly elucidated in medium/large-size laboratory animals. To investigate the impact of ASD in domestic animals, 16 Landrace pigs were exposed to an artificial ASD sandstorm for 6 h. All animals were divided in four cages, and a commercial yellow soil was used for generating artificial mineralogical particles. Blood samples were collected, and necropsies were performed before exposure and 6, 12, 24, and 72 h after exposure. Complete blood cell count and the levels of serum biochemical enzymes, blood gas, electrolytes, and a variety of inflammatory cytokines were evaluated. In addition, histopathological examination was conducted. Various test results proved acute lower airway disorders with systemic inflammation in pigs. To our knowledge, this study is the first to describe experimental research in domestic animals concerning the damage caused by artificial ASD exposure. The results of this study suggest that ASD has importance in terms of not only public health but also of ultimate economic losses in the pork industry.

TNF-α Priming Elicits Robust Immunomodulatory Potential of Human Tonsil-Derived Mesenchymal Stem Cells to Alleviate Murine Colitis.

Mesenchymal stem cells (MSCs) have been spotlighted in the field of cell therapies as a promising tool for the treatment of intractable inflammatory diseases. However, their therapeutic potency still shows a gap between preclinical and clinical settings, and distinctive characteristics of specific tissue-derived MSCs and definitive ways to maximize their beneficial functions have not been fully elucidated yet. We previously identified the unique MSCs population from human palatine tonsil (TMSCs) and revealed their superior properties in proliferation and ROS regulation. Based on these findings, we explored further characteristics of TMSCs particularly focused on immunomodulatory function. We found the merit of TMSCs as a therapeutic agent that retains favorable MSCs properties until relatively late passages and revealed that pre-treatment of TNF-α can enhance the immunomodulatory abilities of TMSCs through the upregulation of the PTGS2/PGE2 axis. TMSCs primed with TNF-α effectively restrained the proliferation and differentiation of T lymphocytes and macrophages in vitro, and more interestingly, these TNF-α-licensed TMSCs exhibited significant prophylactic and therapeutic efficacy in a murine model of autoimmune-mediated acute colitis via clinical and histopathological assessment compared to unprimed naïve TMSCs. These findings provide novel insight into the optimization and standardization of MSCs-based anti-inflammatory therapies, especially targeting inflammatory bowel disease (IBD).

Extracellular Vesicles from SOD3-Transduced Stem Cells Exhibit Improved Immunomodulatory Abilities in the Murine Dermatitis Model.

The immunoregulatory abilities of mesenchymal stem cells (MSCs) have been investigated in various autoimmune and allergic diseases. However, the therapeutic benefits observed in preclinical settings have not been reproducible in clinical trials. This discrepancy is due to insufficient efficacy of MSCs in harsh microenvironments, as well as batch-dependent variability in potency. Therefore, to achieve more beneficial and uniform outcomes, novel strategies are required to potentiate the therapeutic effect of MSCs. One of simple strategies to augment cellular function is genetic manipulation. Several studies showed that transduction of antioxidant enzyme into cells can increase anti-inflammatory effects. Therefore, we evaluated the immunoregulatory abilities of MSCs introduced with extracellular superoxide dismutase 3 (SOD3) in the present study. SOD3-overexpressed MSCs (SOD3-MSCs) reduced the symptoms of murine model of atopic dermatitis (AD)-like inflammation, as well as the differentiation and activation of various immune cells involved in AD progression. Interestingly, extracellular vesicles (EVs) isolated from SOD3-MSCs delivered SOD3 protein. EVs carrying SOD3 also exerted improved therapeutic efficacy, as observed in their parent cells. These results suggest that MSCs transduced with SOD3, an antioxidant enzyme, as well as EVs isolated from modified cells, might be developed as a promising cell-based therapeutics for inflammatory disorders.

The activation of NLRP3 inflammasome potentiates the immunomodulatory abilities of mesenchymal stem cells in a murine colitis model.

Inflammasomes are cytosolic, multiprotein complexes that act at the frontline of the immune responses by recognizing pathogen- or danger-associated molecular patterns or abnormal host molecules. Mesenchymal stem cells (MSCs) have been reported to possess multipotency to differentiate into various cell types and immunoregulatory effects. In this study, we investigated the expression and functional regulation of NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome in human umbilical cord blood-derived MSCs (hUCB-MSCs). hUCB-MSCs expressed inflammasome components that are necessary for its complex assembly. Interestingly, NLRP3 inflammasome activation suppressed the differentiation of hUCB-MSCs into osteoblasts, which was restored when the expression of adaptor proteins for inflammasome assembly was inhibited. Moreover, the suppressive effects of MSCs on T cell responses and the macrophage activation were augmented in response to NLRP3 activation. In vivo studies using colitic mice revealed that the protective abilities of hUCB-MSCs increased after NLRP3 stimulation. In conclusion, our findings suggest that the NLRP3 inflammasome components are expressed in hUCB-MSCs and its activation can regulate the differentiation capability and the immunomodulatory effects of hUCB-MSCs. [BMB Reports 2020; 53(6): 329-334].

Human Tonsil-Derived Mesenchymal Stromal Cells Maintain Proliferating and ROS-Regulatory Properties via Stanniocalcin-1.

Mesenchymal stromal cells (MSCs) from various sources exhibit different potential for stemness and therapeutic abilities. Recently, we reported a unique MSCs from human palatine tonsil (TMSCs) and their superior proliferation capacity compared to MSCs from other sources. However, unique characteristics of each MSC are not yet precisely elucidated. We investigated the role of stanniocalcin-1 (STC1), an anti-oxidative hormone, in the functions of TMSCs. We found that STC1 was highly expressed in TMSC compared with MSCs from bone marrow or adipose tissue. The proliferation, senescence and differentiation of TMSCs were assessed after the inhibition of STC1 expression. STC1 inhibition resulted in a significant decrease in the proliferation of TMSCs and did not affect the differentiation potential. To reveal the anti-oxidative ability of STC1 in TMSCs themselves or against other cell types, the generation of mitochondrial reactive oxygen species (ROS) in TMSC or ROS-mediated production of interleukin (IL)-1ß from macrophage-like cells were detected. Interestingly, the basal level of ROS generation in TMSCs was significantly elevated after STC1 inhibition. Moreover, down-regulation of STC1 impaired the inhibitory effect of TMSCs on IL-1ß production in macrophages. Taken together, these findings indicate that STC1 is highly expressed in TMSCs and plays a critical role in proliferating and ROS-regulatory abilities.

Patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening.

Lung cancer shows substantial genetic and phenotypic heterogeneity across individuals, driving a need for personalised medicine. Here, we report lung cancer organoids and normal bronchial organoids established from patient tissues comprising five histological subtypes of lung cancer and non-neoplastic bronchial mucosa as in vitro models representing individual patient. The lung cancer organoids recapitulate the tissue architecture of the primary lung tumours and maintain the genomic alterations of the original tumours during long-term expansion in vitro. The normal bronchial organoids maintain cellular components of normal bronchial mucosa. Lung cancer organoids respond to drugs based on their genomic alterations: a BRCA2-mutant organoid to olaparib, an EGFR-mutant organoid to erlotinib, and an EGFR-mutant/MET-amplified organoid to crizotinib. Considering the short length of time from organoid establishment to drug testing, our newly developed model may prove useful for predicting patient-specific drug responses through in vitro patient-specific drug trials.

A one-stop microfluidic-based lung cancer organoid culture platform for testing drug sensitivity.

Microfluidic devices as translational research tools provide a potential alternative to animal experiments due to their ability to mimic physiological parameters. Several approaches that can be used to predict the efficacy or toxicity of anticancer drugs are available. In general, standard cell culture systems have the advantages of being relatively cost-effective, having high-throughput capability, and providing convenience. However, these models are inadequate to accurately recapitulate the complex organ-level physiological and pharmacological responses. Here, we present a one-stop microfluidic device enabling both 3-dimensional (3D) lung cancer organoid culturing and drug sensitivity tests directly on a microphysiological system (MPS). Our platform reproducibly yields 3D lung cancer organoids in a size-controllable manner and demonstrates for the first time the production of lung cancer organoids from patients with small-cell lung cancer. Lung cancer organoids derived from primary small-cell lung cancer tumors can rapidly proliferate and exhibit disease-specific characteristics in our MPS. Cisplatin and etoposide, the standard regimen for lung cancer, showed increased apoptosis induction in a concentration-dependent manner, but the organoids contained chemo-resistant cells in the core. We envision that this system may provide important information to guide therapeutic approaches at the preclinical level.

Current Strategies to Enhance Adipose Stem Cell Function: An Update.

Mesenchymal stem cells (MSCs) emerged as a promising therapeutic tool targeting a variety of inflammatory disorders due to their multiple remarkable properties, such as superior immunomodulatory function and tissue-regenerative capacity. Although bone marrow (BM) is a dominant source for adult MSCs, increasing evidence suggests that adipose tissue-derived stem cells (ASCs), which can be easily obtained at a relatively high yield, have potent therapeutic advantages comparable with BM-MSCs. Despite its outstanding benefits in pre-clinical settings, the practical efficacy of ASCs remains controversial since clinical trials with ASC application often resulted in unsatisfactory outcomes. To overcome this challenge, scientists established several strategies to generate highly functional ASCs beyond the naïve cells, including (1) pre-conditioning of ASCs with various stimulants such as inflammatory agents, (2) genetic manipulation of ASCs and (3) modification of culture conditions with three-dimensional (3D) aggregate formation and hypoxic culture. Also, exosomes and other extracellular vesicles secreted from ASCs can be applied directly to recapitulate the beneficial performance of ASCs. This review summarizes the current strategies to improve the therapeutic features of ASCs for successful clinical implementation.

A cell-loss-free concave microwell array based size-controlled multi-cellular tumoroid generation for anti-cancer drug screening.

The 3D multi-cellular tumoroid (MCT) model is an in vivo-like, avascular tumor model that has received much attention as a refined screening platform for drug therapies. Several types of research have been efforted to improve the physiological characteristics of the tumor microenvironment (TME) of the in vivo-like MCTs. Size-controlled MCTs have received much attention for obtaining highly reproducible results in drug screening assays and achieving a homogeneous and meaningful level of biological activities. Here, we describe an effective method for fabricating the size-controlled in vivo-like MCTs using a cell-loss-free (CLF) microwell arrays. The CLF microwell arrays was fabricated by using the simple operation of laser carving of a poly (methyl methacrylate) (PMMA) master mold. We also demonstrated the biophysicochemical effect of tumor microenvironment (TME) resident fibroblasts through the expression of TGFß, αSMA, Type I-, IV collagen, angiogenesis related markers on tumorigenesis, and confirmed the drug response of MCTs with anti-cancer agents. This technology for the fabrication of CLF microwell arrays could be used as an effective method to produce an in vitro tumor model for cancer research and drug discovery.

Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia.

The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.

Regenerative potential of tonsil mesenchymal stem cells on surgical cutaneous defect.

As tissue engineering and regenerative medicine have evolved recently, stem cell therapy has been investigated in the field of impaired wound healing. Several studies have reported that mesenchymal stem cells derived from various tissues including bone marrow and adipose tissue can exert the regenerative efficacy in the wound healing. Previously, we have demonstrated the isolation and characterization of tonsil-derived mesenchymal stem cells (TMSCs) with excellent proliferative property. In the present study, we aimed to evaluate the regenerative efficacy of TMSCs in the wound healing process. Two distinct cutaneous surgical defects were generated in the dorsum of mice. Each wound was treated with TMSCs or phosphate-buffered saline (PBS), respectively. After sacrifice, the skin and subcutaneous tissues around the surgical defect were harvested and assessed for inflammation, re-epithelialization, dermal regeneration, and granulation tissue formation. The administration of TMSCs into wound beds significantly promoted the repair of surgical defects in mice. Especially, TMSCs efficiently contributed to the attenuation of excessive inflammation in the surgical lesion, as well as the augmentation of epidermal and dermal regeneration. To elucidate the underlying mechanisms, TMSCs were analyzed for their potency in immunomodulatory ability on immune cells, stimulatory effect on the proliferation of keratinocytes, and fibroblasts, as well as the regulation of fibroblast differentiation. TMSCs inhibited the non-specific or T-cell-specific proliferation of peripheral blood mononuclear cells, as well as the M1 polarization of macrophage-like cells. Moreover, TMSCs augmented the proliferation of skin-constituting fibroblasts and keratinocytes while they suppressed the differentiation of fibroblasts into myofibroblasts. Taken together, our findings demonstrate the regenerative potential of TMSCs in wound healing process through the regulation on inflammation, proliferation, and remodeling of various skin cells, implying that TMSCs can be a promising alternative for wound repair.

The impact of aging on primate hematopoiesis as interrogated by clonal tracking.

Age-associated changes in hematopoietic stem and progenitor cells (HSPCs) have been carefully documented in mouse models but poorly characterized in primates and humans. To investigate clinically relevant aspects of hematopoietic aging, we compared the clonal output of thousands of genetically barcoded HSPCs in aged vs young macaques after autologous transplantation. Aged macaques showed delayed emergence of output from multipotent (MP) clones, with persistence of lineage-biased clones for many months after engraftment. In contrast to murine aging models reporting persistence of myeloid-biased HSPCs, aged macaques demonstrated persistent output from both B-cell and myeloid-biased clones. Clonal expansions of MP, myeloid-biased, and B-biased clones occurred in aged macaques, providing a potential model for human clonal hematopoiesis of indeterminate prognosis. These results suggest that long-term MP HSPC output is impaired in aged macaques, resulting in differences in the kinetics and lineage reconstitution patterns between young and aged primates in an autologous transplantation setting.