L-plastin associated syndrome of immune deficiency and hematologic cytopenia.

BACKGROUND: LCP1 encodes L-plastin, an actin-bundling protein primarily expressed in hematopoietic cells. In mouse and fish models, LCP1 deficiency has been shown to result in hematologic and immune defects. OBJECTIVE: This study aimed to determine the nature of a human inborn error of immunity resulting from a novel genetic variant of LCP1. METHODS: We performed genetic, protein, and cellular analysis of PBMCs from a kindred with apparent autosomal dominant immune deficiency. We identified a candidate causal mutation in LCP1, which we evaluated by engineering the orthologous mutation in mice and Jurkat cells. RESULTS: A splice-site variant in LCP1 segregated with lymphopenia, neutropenia, and thrombocytopenia. The splicing defect resulted in at least 2 aberrant transcripts, producing an in-frame deletion of 24 nucleotides, and a frameshift deletion of exon 8. Cellular analysis of the kindred revealed a proportionate reduction of T and B cells and a mild expansion of transitional B cells. Similarly, mice carrying the orthologous genetic variant exhibited the same in-frame aberrant transcript, reduced expression Lcp1 and gene dose-dependent leukopenia, mild thrombocytopenia, and lymphopenia, with a significant reduction of T-cell populations. Functional analysis revealed that LCP1c740-1G>A confers a defect in platelet development and function with aberrant spreading on collagen. Immunologic analysis revealed defective actin organization in T cells, reduced migration of PBMCs from patients, splenocytes from mutant mice, and a mutant Jurkat cell line in response to CXCL12; impaired germinal center B-cell expansion after immunization; and reduced cytokinesis during T cell proliferation. CONCLUSIONS: We describe a unique human hematopoietic defect affecting neutrophils, lymphocytes, and platelets arising from partial LCP1 deficiency.

Biomimetic cues from poly(lactic-co-glycolic acid)/hydroxyapatite nano-fibrous scaffolds drive osteogenic commitment in human mesenchymal stem cells in the absence of osteogenic factor supplements.

Mimicking the complex hierarchical architecture of the 'osteon', the functional unit of cortical bone, from the bottom-up offers the possibility of generating mature bone tissue in tissue engineered bone substitutes. In this work, a modular 'bottom-up' approach has been developed to assemble bone niche-mimicking nanocomposite scaffolds composed of aligned electrospun nanofibers of poly(lactic-co-glycolic acid) (PLGA) encapsulating aligned rod-shape nano-sized hydroxyapatite (nHA). By encoding axial orientation of the nHA within these aligned nanocomposite fibers, significant improvements in mechanical properties, surface roughness, hydrophilicity and in vitro simulated body fluid (SBF) mineral deposition were achieved. Moreover, these hierarchical scaffolds induced robust formation of bone hydroxyapatite and osteoblastic maturation of human bone marrow-derived mesenchymal stem cells (hBMSCs) in growth media that was absent of any soluble osteogenic differentiation factors. The results of this investigation confirm that these tailored, aligned nanocomposite fibers, in the absence of media-bone inductive factors, offer the requisite biophysical and biochemical cues to hBMSCs to promote and support their differentiation into mature osteoblast cells and form early bone-like tissue in vitro.

Microbioreactor array screening of Wnt modulators and microenvironmental factors in osteogenic differentiation of mesenchymal progenitor cells.

Cellular microenvironmental conditions coordinate to regulate stem cell populations and their differentiation. Mesenchymal precursor cells (MPCs), which have significant potential for a wide range of therapeutic applications, can be expanded or differentiated into osteo- chondro- and adipogenic lineages. The ability to establish, screen, and control aspects of the microenvironment is paramount if we are to elucidate the complex interplay of signaling events that direct cell fate. Whilst modulation of Wnt signaling may be useful to direct osteogenesis in MPCs, there is still significant controversy over how the Wnt signaling pathway influences osteogenesis. In this study, we utilised a full-factorial microbioreactor array (MBA) to rapidly, combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1) and characterise their effects upon osteogenesis. The MBA screening system showed excellent consistency between donors and experimental runs. CHIR99021 (a Wnt agonist) had a profoundly inhibitory effect upon osteogenesis, contrary to expectations, whilst the effects of the IWP-4 and IWR-1 (Wnt antagonists) were confirmed to be inhibitory to osteogenesis, but to a lesser extent than observed for CHIR99021. Importantly, we demonstrated that these results were translatable to standard culture conditions. Using RT-qPCR of osteogenic and Wnt pathway markers, we showed that CHIR exerted its effects via inhibition of ALP and SPP1 expression, even though other osteogenic markers (RUNX2, MSX2, DLX, COL1A1) were upregulated. Lastly, this MBA platform, due to the continuous provision of medium from the first to the last of ten serially connected culture chambers, permitted new insight into the impacts of paracrine signaling on osteogenic differentiation in MPCs, with factors secreted by the MPCs in upstream chambers enhancing the differentiation of cells in downstream chambers. Insights provided by this cell-based assay system will be key to better understanding signaling mechanisms, as well as optimizing MPC growth and differentiation conditions for therapeutic applications.