Efficient In Vitro Generation of IL-22-Secreting ILC3 From CD34+ Hematopoietic Progenitors in a Human Mesenchymal Stem Cell Niche

Innate lymphoid cells (ILCs) and in particular ILC3s have been described to be vital for mucosal barrier functions and homeostasis within the gastrointestinal (GI) tract. Importantly, IL-22-secreting ILC3 have been implicated in the control of inflammatory bowel disease (IBD) and were shown to reduce the incidence of graft-versus-host disease (GvHD) as well as the risk of transplant rejection. Unfortunately, IL-22-secreting ILC3 are primarily located in mucosal tissues and are not found within the circulation, making access to them in humans challenging. On this account, there is a growing desire for clinically applicable protocols for in vitro generation of effector ILC3. Here, we present an approach for faithful generation of functionally competent human ILC3s from cord blood-derived CD34+ hematopoietic progenitors on layers of human mesenchymal stem cells (MSCs) generated in good manufacturing practice (GMP) quality. The in vitro-generated ILC3s phenotypically, functionally, and transcriptionally resemble bona fide tissue ILC3 with high expression of the transcription factors (TF) RorγT, AHR, and ID2, as well as the surface receptors CD117, CD56, and NKp44. Importantly, the majority of ILC3 belonged to the desired effector subtype with high IL-22 and low IL-17 production. The protocol thus combines the advantages of avoiding xenogeneic components, which were necessary in previous protocols, with a high propensity for generation of IL-22-producing ILC3. The present approach is suitable for the generation of large amounts of ILC3 in an all-human system, which could facilitate development of clinical strategies for ILC3-based therapy in inflammatory diseases and cancer

A robust method to analyze copy number alterations of less than 100 kb in single cells using oligonucleotide array CGH.

Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data

aCGH experiments with CellSearch® identified and MoFlo™ XDP sorted WGAM single cells from cell line OE19.

<p>A) Screenshot from CellSearch® System from an EpCAM captured CK⁺/DAPI⁺/CD45⁻ tumor cell: overlay of DAPI⁺/CK-PE⁺ (upper left), CK-PE⁺ (upper right), DAPI⁺ (lower left)/CD45-APC⁻ (lower right). B) & C) Dotplot from MoFlo™ XDP, CK⁺ and CD45⁺ cells (B) and DAPI⁺ cells (C). D) Overview of the genomewide profiles from two cells identified with CellSearch®, isolated with MoFlo™ XDP and amplified by WGAM. Black =  gDNA, blue = WGAM single cell #1 and red = WGAM single cell #2.</p

Uptake dynamics of graphene quantum dots into primary human blood cells following in vitro exposure

Human leukocytes obtained from samples of leukapheresis products of three healthy donors stimulated by granulocyte colony stimulating factor (G-CSF) were exposed to graphene quantum dots. A time- and concentration dependent uptake was observed with a significantly greater uptake into monocytes and granulocytes in comparison to lymphocytes, suggesting a better incorporation ability of cells with phagocytotic properties. The uptake rates also correlate with the cell membrane area. Looking at the different lymphoid subsets a greater uptake was found into CD19+ B-, CD56+ natural killer cells and CD34+ hematopoietic stem cells (HSC) in comparison to CD4+ T- and CD8+ T cells. Independent of the cell type studied, the observed uptake dynamics is consistent with a diffusion-driven process, which allows the determination of cell-specific membrane permeabilities for the graphene quantum dots. The toxicity of the quantum dots is relatively low resulting in a 90% viability of the entire leukocyte population after 36 hours of exposure to GQDs at a concentration of 500 μg ml−1

ROC curves for aCGH experiments with WGAM amplified single cells.

<p>Cell lines OE19 A) & B) and REH C) & D). An experiment performed according to standard protocol and with gDNA from each corresponding cell line served as a reference array for ROC analysis. A) Digested high molecular DNA vs. single cell amplified DNA used as reference DNA. B) Comparison of different labeling methods (RP = random-primed labeling, MSE = MSE-PCR based labeling 1 or 2, TS = Thermosequenase labeling, ULS = Universal Linkage System™). C) Comparison of aCGH platforms (2×400k, MPS = 5.3 kb, 4×180k, MPS = 13 kb and 8×60k, MPS = 41.4 kb). D) Comparison of different amplification methods (WGAM, WGAN and WGAS).</p

Visualization of smallest detected alterations.

<p>Smallest alteration (blue area) in gDNA and single cell amplified DNA generated with three WGA methods (WGAM, WGAN & WGAS from left to right). A) 56 kb deletion of material from chromosome 7p14.1 in cell line REH. Note that the deletion could not be retrieved in the WGAS amplified single cell. B) 115 kb gain of material from chromosome 17q12 in the cell line OE19.</p

Effect of custom aberration filter.

<p>Visualization of chromosomal alterations on chromosome 1 in Genomic Workbench using aberration detection algorithm ADM-2. Blue filled areas denote a gain (right) or loss (left) of chromosomal material. A) Analysis of gDNA, WGAM-, WGAN- and WGAS-single cell amplified DNA without aberration filter. B) Analysis of the same samples with aberration filters (≥3 consecutive oligonucleotides, ≥ log2ratio ±0.25).</p

Genome wide aCGH profiles of gDNA and associated amplified single cell DNA from healthy controls, REH and OE19.

<p>Peaks upstream the baseline (red area) denote a gain, peaks downstream the baseline (green area) indicate a loss of chromosomal material. A) Healthy control black line = gDNA, red line = female single cell vs. male single cell, blue line = male vs. female single cell. B) REH black line = gDNA, red = single cell. C) OE19 black line = gDNA, red = single cell.</p

DataSheet_1_Efficient In Vitro Generation of IL-22-Secreting ILC3 From CD34+ Hematopoietic Progenitors in a Human Mesenchymal Stem Cell Niche.pdf

Innate lymphoid cells (ILCs) and in particular ILC3s have been described to be vital for mucosal barrier functions and homeostasis within the gastrointestinal (GI) tract. Importantly, IL-22-secreting ILC3 have been implicated in the control of inflammatory bowel disease (IBD) and were shown to reduce the incidence of graft-versus-host disease (GvHD) as well as the risk of transplant rejection. Unfortunately, IL-22-secreting ILC3 are primarily located in mucosal tissues and are not found within the circulation, making access to them in humans challenging. On this account, there is a growing desire for clinically applicable protocols for in vitro generation of effector ILC3. Here, we present an approach for faithful generation of functionally competent human ILC3s from cord blood-derived CD34+ hematopoietic progenitors on layers of human mesenchymal stem cells (MSCs) generated in good manufacturing practice (GMP) quality. The in vitro-generated ILC3s phenotypically, functionally, and transcriptionally resemble bona fide tissue ILC3 with high expression of the transcription factors (TF) RorγT, AHR, and ID2, as well as the surface receptors CD117, CD56, and NKp44. Importantly, the majority of ILC3 belonged to the desired effector subtype with high IL-22 and low IL-17 production. The protocol thus combines the advantages of avoiding xenogeneic components, which were necessary in previous protocols, with a high propensity for generation of IL-22-producing ILC3. The present approach is suitable for the generation of large amounts of ILC3 in an all-human system, which could facilitate development of clinical strategies for ILC3-based therapy in inflammatory diseases and cancer.</p