Pulmonary Transplantation of Human Induced Pluripotent Stem Cell-derived Macrophages Ameliorates Pulmonary Alveolar Proteinosis.

RATIONALE: Although the transplantation of induced pluripotent stem cell (iPSC)-derived cells harbors enormous potential for the treatment of pulmonary diseases, in vivo data demonstrating clear therapeutic benefits of human iPSC-derived cells in lung disease models are missing. OBJECTIVES: We have tested the therapeutic potential of iPSC-derived macrophages in a humanized disease model of hereditary pulmonary alveolar proteinosis (PAP). Hereditary PAP is caused by a genetic defect of the GM-CSF (granulocyte-macrophage colony-stimulating factor) receptor, which leads to disturbed macrophage differentiation and protein/surfactant degradation in the lungs, subsequently resulting in severe respiratory insufficiency. METHODS: Macrophages derived from human iPSCs underwent intrapulmonary transplantation into humanized PAP mice, and engraftment, in vivo differentiation, and therapeutic efficacy of the transplanted cells were analyzed. MEASUREMENTS AND MAIN RESULTS: On intratracheal application, iPSC-derived macrophages engrafted in the lungs of humanized PAP mice. After 2 months, transplanted cells displayed the typical morphology, surface markers, functionality, and transcription profile of primary human alveolar macrophages. Alveolar proteinosis was significantly reduced as demonstrated by diminished protein content and surfactant protein D levels, decreased turbidity of the BAL fluid, and reduced surfactant deposition in the lungs of transplanted mice. CONCLUSIONS: We here demonstrate for the first time that pulmonary transplantation of human iPSC-derived macrophages leads to pulmonary engraftment, their in situ differentiation to an alveolar macrophage phenotype, and a reduction of alveolar proteinosis in a humanized PAP model. To our knowledge, this finding presents the first proof-of-concept for the therapeutic potential of human iPSC-derived cells in a pulmonary disease and may have profound implications beyond the rare disease of PAP.

Immunophenotyping of cerebrospinal fluid cells by Chipcytometry.

BACKGROUND: The gold standard in cerebrospinal fluid (CSF) cell immunophenotyping is flow cytometry. Nevertheless, the small amount of CSF cells and the invasive character of lumbar puncture limit the spectrum of possible investigation. Chipcytometry, a modified approach to slide-based cytometry, might be a useful tool for CSF analysis due to the possibility of iterative staining, imaging, and bleaching cycles. The aim of this study was to compare flow cytometric leukocyte subset analysis with Chipcytometry comparing the percentage distribution of distinct cell populations and the T-cell CD4:CD8 ratio. Moreover, this study investigated the interpretability of chips loaded with CSF cells and examined the applicability of Chipcytometry in clinical practice. METHODS: 375 CSF samples from 364 patients were analyzed by Chipcytometry using an automated upright microscope. Cell surface molecules were stained using fluorescence-labeled monoclonal antibodies. For cross-validation experiments, flow cytometry data of six patients were analyzed and matched with Chipcytometry data. RESULTS: Our experiments showed a better agreement examined by Bland-Altman analysis for samples with CSF pleocytosis than for normocellular CSF samples. Data were more consistent for B cells and CD4:CD8 ratio than for T cells and monocytes. Advantages of Chipcytometry compared to flow cytometry are that cells once fixated can be analyzed for up to 20 months with additional markers at any time. The clinical application of Chipcytometry is demonstrated by two illustrative case reports. However, the low amount of CSF cells limits the analysis of normocellular CSF samples, as in our cohort only 11.7% of respectively loaded chips had sufficient cell density for further investigation compared to 59.8% of all chips loaded with samples with elevated cell counts (≥ 5/µl). Varying centrifuge settings, tube materials and resuspension technique were not able to increase the cell yield. CONCLUSION: In summary, the results demonstrate the great potential of Chipcytometry of CSF cells for both scientific questions and routine diagnostic. A new chip design optimized to meet the requirements of CSF would greatly enhance the value of this method. Cross-validation results need to be confirmed in a larger cohort.

In situ detection of CD73+ CD90+ CD105+ lineage: Mesenchymal stromal cells in human placenta and bone marrow specimens by chipcytometry.

Mesenchymal stromal cells (MSCs) support endogenous regeneration and present therefore promising opportunities for in situ tissue engineering. They can be isolated and expanded from various tissues, for example, bone marrow, adipose tissue, or placenta. The minimal consensus definition criteria of ex vivo expanded MSCs requires them to be positive for CD73, CD90, and CD105 expression, while being negative for CD34, CD45, CD14, CD19, and HLA-DR. This study aimed to compare the in situ phenotype of MSCs with that of their culture-expanded progeny. We report for the first time in situ detection of cells expressing this marker combination in human placenta cryosections as well as in bone marrow aspirates using multiplex-immunohistology (Chipcytometry), a technique that allows staining of more than 100 biomarkers consecutively on the same cell. © 2018 International Society for Advancement of Cytometry.

A nano-enabled cancer-specific ITCH RNAi chemotherapy booster for pancreatic cancer.

Gemcitabine is currently the standard therapy for pancreatic cancer. However, growing concerns over gemcitabine resistance mean that new combinatory therapies are required to prevent loss of efficacy with prolonged treatment. Here, we suggest that this could be achieved through co-administration of RNA interference agents targeting the ubiquitin ligase ITCH. Stable anti-ITCH siRNA and shRNA dendriplexes with a desirable safety profile were prepared using generation 3 poly(propylenimine) dendrimers (DAB-Am16). The complexes were efficiently taken up by human pancreatic cancer cells and produced a 40-60% decrease in ITCH RNA and protein expression in vitro (si/shRNA) and in a xenograft model of pancreatic cancer (shRNA). When co-administered with gemcitabine (100 mg/kg/week) at a subtherapeutic dose, treatment with ITCH-shRNA (3x 50 mg/week) was able to fully suppress tumour growth for 17 days, suggesting that downregulation of ITCH mediated by DAB-Am16/shRNA sensitizes pancreatic cancer to gemcitabine in an efficient and specific manner. FROM THE CLINICAL EDITOR: Gemcitabine delivery to pancreatic cancer often results in the common problem of drug resistance. This team overcame the problem through co-administration of siRNA and shRNA dendriplexes targeting the ubiquitin ligase ITCH.

A preliminary study for the assessment of PD-L1 and PD-L2 on circulating tumor cells by microfluidic-based chipcytometry.

AIM: Expression of PD-L1 in the tumor is associated with more favorable responses to anti-PD-1 therapy in multiple cancers. However, obtaining tumor biopsies for PD-L1 interrogation is an invasive procedure and challenging to assess repeatedly as the disease progresses. MATERIALS & METHODS: Here we assess an alternative, minimally invasive approach to analyze blood samples for circulating tumor cells (CTCs) that have broken away from the tumor and entered the periphery. Our approach uses sized-based microfluidic CTC enrichment and subsequent characterization with microfluidic-based cytometry (chipcytometry). CONCLUSION: We demonstrate tumor-cell detection and characterization for PD-L1, and other markers, in both spiked and patient samples. This preliminary communication is the first report using chipcytometry for the characterization of CTCs.

Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis.

Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor genes, resulting in disturbed alveolar macrophage differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. So far, the only effective treatment for herPAP is repetitive whole-lung lavage, a merely symptomatic and highly invasive procedure. We introduce pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for herPAP. In a murine disease model, intrapulmonary transplanted macrophage progenitors displayed selective, long-term pulmonary engraftment and differentiation into functional alveolar macrophages. A single transplantation ameliorated the herPAP phenotype for at least 9 months, resulting in significantly reduced alveolar proteinosis, normalized lung densities in chest computed tomography, and improved lung function. A significant and sustained disease resolution was also observed in a second, humanized herPAP model after intrapulmonary transplantation of human macrophage progenitors. The therapeutic effect was mediated by long-lived, lung-resident macrophages, which displayed functional and phenotypical characteristics of primary human alveolar macrophages. Our findings present the concept of organotopic transplantation of macrophage progenitors as an effective and long-lasting therapy of herPAP and may also serve as a proof of principle for other diseases, expanding current stem cell-based strategies toward potent concepts using the transplantation of differentiated cells.