Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions

<div><p>Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3×10⁶±0.3×10⁶ floating monocytes from approximately 30 clusters of ESCs/iPSCs 5–6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.</p> </div

Functional assays for M1/M2 macrophages derived from pluripotent stem cells.

<p>(A) Flow cytometric analysis of M1/M2 macrophages derived from pluripotent stem cells. (B) The levels of IL-12p70 and IL-10 in supernatants of culture medium with M1/M2 macrophages derived from pluripotent stem cells 24 hours after LPS stimulation. The data of KhES1-derived cells are shown as representative.</p

Phenotype analysis and gene expression pattern of monocytic lineage cells derived from pluripotent stem cells.

<p>(A) Flow cytometric analysis of monocytic lineage cells derived sequentially from pluripotent stem cells. An analysis of adherent cells on day 6 and supernatant cells on day 13 and 18 is shown. (B) May-Giemsa staining of CD14⁺ monocyte-like cells derived from KhES1 on day 16 (left) and primary human monocytes (right). (C) Esterase staining for CD14⁺ monocyte-like cells derived from KhES1 on day 16. (D) The percentage of CD14⁺ cells within the total floating cells derived from KhES1/iPS-201B7 was evaluated from day 13 to day 28. (E) May-Giemsa staining (left) and phase contrast image (right) of mature DCs derived from pluripotent stem cells. (F) Flow cytometric analysis of immature/mature DCs derived from pluripotent stem cells. (G) Phase contrast image and flow cytometric analysis of macrophages derived from pluripotent stem cells.(H) RT-PCR analysis of monocytic lineage cells derived from KhES1/iPS-201B7 clones for expression of monocytic lineage marker genes (<i>PU.1, c-MAF, TLR4, CCL17</i> and <i>CCL18</i>). Peripheral blood monocytes and peripheral blood monocyte-derived mature DCs were used as positive controls.(A–C, E–G) The data from KhES1-derived cells are shown as representative.</p

Functional assays for monocytes derived from pluripotent stem cells.

<p>(A) The levels of IL-6 and TNFα in supernatants of PS-Mo culture medium 4 hours after LPS stimulation. The levels of IL-1β were measured 4 hours after LPS stimulation with/without an additional 30-minute ATP stimulation. (B) Flow cytometric analysis of CX3CR1 on PS-Mo. (C) Chemotaxis assay of PS-Mo for CX3CL1 (fractalkine) using a trans-well migration assay. After the addition of CX3CL1 into either the bottom or top of the trans-well chamber, PS-Mo were applied and incubated for 5 hours at 37°C. (D) Antigen uptake was evaluated in monocytes, immature DCs and mature DCs derived from pluripotent stem cells by examining the fluorescence intensity of Alexa fluor 488-conjugated ovalbumin 45 minute after incubation at 37°C (black). Control samples (white) were kept on ice. (B–D) The data of KhES1-derived cells are shown as representative. PS-Mo: monocyte derived from pluripotent stem cells.</p

Functional assays for dendritic cells derived from pluripotent stem cells.

<p>(A) Flow cytometric analysis of immature/mature DCs derived from pluripotent stem cells. (B) The levels of IL-10 and TNFα in supernatants of culture medium with PS-DCs 24 hours after LPS stimulation. (C) The proliferation of allogeneic naïve T cells (1×10⁵ cells per well) co-cultured with 40 Gy-irradiated stimulator cells for 3 days was evaluated. The proliferation of naïve T cells in the last 16 hours was measured by ³H-thymidine uptake. (A–C) The data of KhES1-derived cells are shown as representative.</p

DataSheet_1_Assessment of type I interferon signatures in undifferentiated inflammatory diseases: A Japanese multicenter experience.pdf

PurposeUpregulation of type I interferon (IFN) signaling has been increasingly detected in inflammatory diseases. Recently, upregulation of the IFN signature has been suggested as a potential biomarker of IFN-driven inflammatory diseases. Yet, it remains unclear to what extent type I IFN is involved in the pathogenesis of undifferentiated inflammatory diseases. This study aimed to quantify the type I IFN signature in clinically undiagnosed patients and assess clinical characteristics in those with a high IFN signature.MethodsThe type I IFN signature was measured in patients’ whole blood cells. Clinical and biological data were collected retrospectively, and an intensive genetic analysis was performed in undiagnosed patients with a high IFN signature.ResultsA total of 117 samples from 94 patients with inflammatory diseases, including 37 undiagnosed cases, were analyzed. Increased IFN signaling was observed in 19 undiagnosed patients, with 10 exhibiting clinical features commonly found in type I interferonopathies. Skin manifestations, observed in eight patients, were macroscopically and histologically similar to those found in proteasome-associated autoinflammatory syndrome. Genetic analysis identified novel mutations in the PSMB8 gene of one patient, and rare variants of unknown significance in genes linked to type I IFN signaling in four patients. A JAK inhibitor effectively treated the patient with the PSMB8 mutations. Patients with clinically quiescent idiopathic pulmonary hemosiderosis and A20 haploinsufficiency showed enhanced IFN signaling.ConclusionsHalf of the patients examined in this study, with undifferentiated inflammatory diseases, clinically quiescent A20 haploinsufficiency, or idiopathic pulmonary hemosiderosis, had an elevated type I IFN signature.</p