A Novel Secretome Biotherapeutic Influences Regeneration in Critical Size Bone Defects.

Severe traumatic injuries often result in critical size bone defects, which are unable to heal without treatment. Autologous grafting is the standard of care but requires additional surgeries for graft procurement. Amnion-derived multipotent progenitor cells release a secretome of biomolecules identified as integral to the process of bone regeneration and angiogenesis. This secretome is currently under development as a biotherapeutic. The efficacy of this secretome biotherapeutic was evaluated in vitro on the proliferation and migration of mesenchymal stem cells and osteoprogenitor cells as well as in vivo using a critical size rat calvarial defect model. The secretome biotherapeutic was loaded onto a collagen scaffold and placed into the defect, which was allowed to heal for 4 and 12 weeks. The secretome biotherapeutic enhanced the proliferation and migration of mesenchymal stem cells and proliferation of osteoprogenitor cells. Further, the secretome biotherapeutic improved new bone volume and connectivity by 12 weeks and significantly improved angiogenesis at 4 weeks and bone density at 4 and 12 weeks with no deleterious effects. The improvement in new bone volume, connectivity, and angiogenesis suggests that the secretome biotherapeutic has beneficial effects for bone healing and a higher dose of the secretome biotherapeutic may further improve regeneration.

Amnion-derived multipotent progenitor cells inhibit blood monocyte differentiation into mature dendritic cells.

Cells derived from the placenta have become the focus of extensive research concerning their ability to be used for regenerative medicine or cellular therapies. In a previous study, we characterized amnion-derived multipotent progenitor cells, or AMP cells, by in vitro methods and showed they were able to inhibit antigen-specific T-cell proliferation in a cell-to-cell contact-dependent fashion. Here we examine specific mechanisms involved in immunomodulation by AMP cells. We found that AMP cells significantly inhibited monocyte-derived myeloid dendritic cell (DC) maturation when placed in coculture. Cocultured monocytes retained the nondifferentiated macrophage marker CD14 while exhibiting significant reduction in DC maturation markers CD83 and CD1a, indicating an immature DC maturation state that is pivotal in determining its immune stimulatory or regulatory status. This effect was again dependent on cell-to-cell contact interaction. We also found a significant shift in cytokines present in the microenvironment of cocultures, which indicated a regulatory DC function rather than a stimulatory cell type. Here supernatants taken from AMP cell/monocyte cocultures yielded significant levels of regulatory cytokines, such as PGE2, IL-6, IL-10, and MIC-1. The soluble form of HLA-G was also found at higher levels in cocultures. In contrast, supernatants contained significantly less amounts of the T-cell-stimulating factor IL-12, which is normally produced by activated DCs. Interestingly, cocultured monocytes acquired significant expression of HLA-G on their cell surface over time. HLA-G has multifaceted immunological implications and may be a key molecule in influencing these cells to behave as regulatory DCs. Together, the influence of AMP cells on maturing DCs may favor a regulatory pathway that can be useful for therapeutic applications for immune-mediated disorders or transplantation therapies.

Immunogenicity and immunomodulatory effects of amnion-derived multipotent progenitor cells.

This is the first study on the immunologic properties of a clinically relevant population of cells derived from the amnion of human placenta. Unlike other cells from the amnion, these amnion-derived multipotent progenitor cells (AMP cells), from human amnion, grow in serum-free conditions and have never been cultured in the presence of medium containing animal-derived components. This study reports the immunologic characteristics of AMP cells and their roles as immunomodulators. Characterization of AMP cells revealed the presence of major histocompatibility complex (MHC) class I but the lack of class II antigens and absence of co-stimulatory molecules B7-1 and B7-2. The nonclassical human leukocyte antigen (HLA)-G was expressed at low levels on cultured AMP cells. Expression was significantly increased after interferon-gamma (IFN-gamma) treatment. Cultured peripheral blood mononuclear cells did not respond to irradiated AMP cells, indicated by lack of proliferation as measured by standard mixed lymphocyte reaction. Culturing AMP cells with IFN-gamma did not reverse this result and did not upregulate class II expression. The AMP cells were shown to have immunomodulatory capabilities by inhibiting peripheral blood mononuclear cell proliferative responses to mitogen, alloantigen, and recall antigen, but the AMP cells were unable to inhibit preactivated T-cell blast response to growth factor media. This immunomodulatory effect of AMP cells was found to be dependent on cell-to-cell contact.

Real time PCR assays to detect common mutations in the biotinidase gene and application of mutational analysis to newborn screening for biotinidase deficiency.

Biotinidase deficiency is an autosomal recessive disorder of biotin metabolism caused by defects in the biotinidase gene. Symptoms of biotinidase deficiency are resolved or prevented with oral biotin supplementation and as such newborn screening is performed to prospectively identify affected individuals prior to the onset of symptoms. Biotinidase deficiency is detected by determining the activity of the biotinidase enzyme utilizing the newborn dried blood spot and colorimetric end point analysis. While newborn screening by enzyme analysis is effective, external factors may compromise results of the enzyme analysis and difficulty is encountered in distinguishing between complete and partial enzyme deficiencies. In the United States, the four mutations most commonly associated with complete biotinidase deficiency are c98:d7i3, Q456H, R538C, and the double mutation D444H:A171T. Partial biotinidase deficiency is almost universally attributed to the D444H mutation. To more effectively distinguish between profound and partial biotinidase deficiency, a panel of assays utilizing real time PCR and melting curve analysis using Light Cycler technology was developed. Employing DNA extracted from the original dried blood specimens from newborns identified through prospective newborn screening as presumptive positive for biotinidase deficiency, the specimens were analyzed for the presence of the five common mutations. Using this approach it was possible to separate newborns with partial and complete deficiency from each other as well as from many of those with false positive results. In most cases it was also possible to correlate the genotype with the degree of residual enzyme activity present. In newborn screening for biotinidase deficiency, we have shown that the analysis of common mutations is useful in distinguishing between partial and complete enzyme deficiency as well as improving specificity. Combining biotinidase enzyme analysis with genotypic data also increases the sensitivity of screening for biotinidase deficiency and provides information useful to clinicians earlier than would otherwise be possible.

Analysis of common mutations in the galactose-1-phosphate uridyl transferase gene: new assays to increase the sensitivity and specificity of newborn screening for galactosemia.

Classical galactosemia is a genetic disease caused by mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. Prospective newborn screening for galactosemia is routine and utilizes the universally collected newborn dried blood specimen on filter paper. Screening for galactosemia is achieved through analysis of total galactose (galactose and galactose-1-phosphate) and/or determining the activity of the GALT enzyme. While this approach is effective, environmental factors and the high frequency of the Duarte D2 mutation (N314D) does lead to false positive results. Using DNA derived from the original newborn dried blood specimen and Light Cycler technology a panel of five assays able to detect the four most frequently encountered classical galactosemia alleles (Q188R, S135L, K285N, and L195P) and the N314D Duarte variant mutation are described. The five assays are performed simultaneously using common conditions. Including DNA preparation, set-up, amplification, and analysis the genotype data for all five loci is obtained in less than 2 hours. The assays are easily interpreted and amenable to high-throughput newborn screening. Mutational analysis is useful to reduce false positive results, differentiate D/G mixed heterozygotes from classical galactosemia, and to clearly identify a very high percentage of those affected by classical galactosemia.