iPSC-Based Modeling of RAG2 Severe Combined Immunodeficiency Reveals Multiple T Cell Developmental Arrests.

RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7-CD5- to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7-CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks.

Identification of receptor-type protein tyrosine phosphatase µ as a new marker for osteocytes.

Osteocytes are the predominant cells in bone, where they form a cellular network and display important functions in bone homeostasis, phosphate metabolism and mechanical transduction. Several proteins strongly expressed by osteocytes are involved in these processes, e.g., sclerostin, DMP-1, PHEX, FGF23 and MEPE, while others are upregulated during differentiation of osteoblasts into osteocytes, e.g., osteocalcin and E11. The receptor-type protein tyrosine phosphatase µ (RPTPµ) has been described to be expressed in cells which display a cellular network, e.g., endothelial and neuronal cells, and is implied in mechanotransduction. In a capillary outgrowth assay using metatarsals derived from RPTPµ-knock-out/LacZ knock-in mice, we observed that the capillary structures grown out of the metatarsals were stained blue, as expected. Surprisingly, cells within the metatarsal bone tissue were positive for LacZ activity as well, indicating that RPTPµ is also expressed by osteocytes. Subsequent histochemical analysis showed that within bone, RPTPµ is expressed exclusively in early-stage osteocytes. Analysis of bone marrow cell cultures revealed that osteocytes are present in the nodules and an enzymatic assay enabled the quantification of the amount of osteocytes. No apparent bone phenotype was observed when tibiae of RPTPµ-knock-out/LacZ knock-in mice were analyzed by µCT at several time points during aging, although a significant reduction in cortical bone was observed in RPTPµ-knock-out/LacZ knock-in mice at 20 weeks. Changes in trabecular bone were more subtle. Our data show that RPTPµ is a new marker for osteocytes.

Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a classical BMP antagonist.

Sclerosteosis, a skeletal disorder characterized by high bone mass due to increased osteoblast activity, is caused by loss of the SOST gene product, sclerostin. The localization in bone and the mechanism of action of sclerostin are not yet known, but it has been hypothesized that it may act as a bone morphogenetic protein (BMP) antagonist. We show here that SOST/sclerostin is expressed exclusively by osteocytes in mouse and human bone and inhibits the differentiation and mineralization of murine preosteoblastic cells (KS483). Although sclerostin shares some of the actions of the BMP antagonist noggin, we show here that it also has actions distinctly different from it. In contrast to noggin, sclerostin did not inhibit basal alkaline phosphatase (ALP) activity in KS483 cells, nor did it antagonize BMP-stimulated ALP activity in mouse C2C12 cells. In addition, sclerostin had no effect on BMP-stimulated Smad phosphorylation and direct transcriptional activation of MSX-2 and BMP response element reporter constructs in KS483 cells. Its unique localization and action on osteoblasts suggest that sclerostin may be the previously proposed osteocyte-derived factor that is transported to osteoblasts at the bone surface and inhibits bone formation.