Multi-lineage reconstitution in adult NSG mice.

<p>Sub-lethally irradiated NSG adults were injected with 2.5–5×10⁵ expanded CD34⁺ CD133⁺ cells. Cells in the bone marrow (A–E, L), blood (F, O), thymus (M) and spleen (G–J, K, N) were analyzed by flow cytometry 12 weeks after reconstitution. Shows are staining profiles for erythrocytes (CD235ab⁺) and platelets (CD41⁺) (A and F), total leukocytes (human CD45⁺) (B and G), myelomonocytes (CD14⁺ CD33⁺) (C and H), dendritic cells (CD11c⁺ CD209⁺) (D and I), granulocytes (CD15⁺) and natural killer cells (CD56⁺) (E and J), B cell stages (proB - CD34⁺ CD10⁺, immature - CD10⁺, mature - IgM⁺ IgD⁺) (K and L), T cell precursors (double negative, double positive and single positive) (M) and mature T cells (CD45⁺ CD3⁺ expressing either CD4 or CD8) (N and O). The numbers indicate percentages of cells in the gated region. Representative data from one of at least 5 mice are shown. All cells shown are gated on live human cells (FSC/SSC/live/CD45⁺) except A, F (FSC/SSC only) B, G (FSC/SSC/live) and the IgM vs IgG stains (FSC/SSC/live/CD45⁺/CD19⁺). Summaries of human cell characterization can also be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018382#pone-0018382-t002" target="_blank">Table 2</a>.</p

Summary of secondary reconstitutions in NSG mice.

<p>2.5–5×10⁵ expanded CD34⁺ CD133⁺ cells were transferred into the primary neonate or adult NSG recipients. 14–28 weeks after transfer, single cell suspension was prepared from the bone marrow of recipient mice. Human cells were isolated by either depleting mouse CD45⁺ cells or enriching for human CD34⁺ cells and then transferred into secondary neonate or adult recipients. Each secondary recipient received an equivalent of one femur's worth of enriched human cells. The numbers of CD34⁺ cells were calculated and shown to the nearest 10,000 cells. n.d. – not determined. 12 weeks after secondary transfer, chimerism was assessed in the bone marrow by staining for human and mouse CD45.</p

Expanded CD34⁺ CD133⁺ cells are SCID repopulating cells.

<p>(A) Limiting dilution analysis comparing cultured (squares) vs. uncultured (circles) cells for SRC frequency. CD133⁺ cord blood cells (5,000, 1,000 and 400) and their cultured progeny were injected into sub-lethally irradiated neonate recipients. Recipients were analyzed for human cell reconstitution in the bone marrow by staining for human CD45 and mouse CD45 followed by flow cytometry. SRC frequencies calculated by Poisson statistics are shown for each population based on number of input CD34⁺ CD133⁺ cells. Values for a 0.5% cutoff (top) and 0.1% cutoff (bottom) are shown. (B) Representative CD34 versus CD133 staining profiles of purified human HSCs at the start of in vitro expansion and cells after culture for 10 days. The number indicates percentage of CD34⁺ CD133⁺ cells in the gated region. (C) Comparison of bone marrow chimerism (human CD45 vs. mouse CD45.1) in sub-lethally irradiated neonate NSG recipients injected with 5000, 1000 and 400 cultured or uncultured CD34⁺ CD133⁺ cells. Horizontal bars show the mean, each symbol is an individual mouse. Two tail ANOVA testing shows no significant differences between data sets (p = 0.23). (D) Comparison of reconstitution in neonate NSG recipients transferred with 100,000 purified, cultured CD34⁺ CD133⁺ cells or total cultured cells (unpurified) containing 100,000 CD34⁺ CD133⁺ cells. Peripheral blood mononuclear cells were analyzed for human and mouse CD45 12 weeks after transfer. Mean ± SD is shown with 7 mice per group. (E) Limiting dilution analysis comparing purified (circles) and unpurified (squares) CD34⁺ CD133⁺ cells for SRC frequency. 300 and 1000 purified CD34⁺ CD133⁺ cells or total cells containing 300 and 1000 CD34⁺ CD133⁺ cells were transferred into sub-lethally irradiated adult NSG mice. Recipients were analyzed for human cell reconstitution in the bone marrow 8 weeks later by flow cytometry. Frequencies calculated using Poisson statistics are shown for each population based on the number of CD34⁺ CD133⁺ cells. All limiting dilutions represent 6–10 mice per data point and are single cultures representative of at least 2 independent experiments.</p

Expanded CD34⁺ CD133⁺ cells give robust reconstitution in both neonate and adult recipients.

<p>(A) Comparison of reconstitution levels in mice injected with different numbers of expanded cells. Sub-lethally irradiated adult NSG recipients were injected with cultured cells containing 0.5×10⁵, 2.5×10⁵ or 5×10⁵ CD34⁺ CD133⁺ cells. Eight weeks later, PBMCs were analyzed for human and mouse CD45 expression. Mean ± SD is shown, n = 6–8 per group. (B) Comparison of reconstitution levels in the bone marrow of neonate and adult recipients. Sub-lethally irradiated adult NSG recipients were injected with expanded cells containing 2.5×10⁵ CD34⁺ CD133⁺ cells. Sub-lethally irradiated neonates were injected with 1×10⁵ unexpanded or expanded CD34⁺ CD133⁺ cells. Twelve to sixteen week later, bone marrow was analyzed for human and mouse CD45 expression. Mean ± SD is shown, n = 4–6 per group.</p

Summary of reconstitution of adult NSG mice injected with 2.5–5×10⁵ expanded CD34⁺CD133⁺cells.

<p>Data are a compilation of 24 mice. Each stain was performed on a minimum of 4 mice and all stains were performed on mice derived from at least two unrelated cords. All percentage calculations are based on the following gated groups: human leukocytes on total live (DAPI/PI negative) human or mouse CD45⁺ cells, erythrocytes and platelets on FSC versus SSC gate, and all other groups on live human CD45⁺ cells. Shown are mean percentage and SD of specific cell types in blood, spleen and bone marrow. ND, not determined.</p

Graphene Multilayers as Gates for Multi-Week Sequential Release of Proteins from Surfaces

The ability to control the timing and order of release of different therapeutic drugs will play a pivotal role in improving patient care and simplifying treatment regimes in the clinic. The controlled sequential release of a broad range of small and macromolecules from thin film coatings offers a simple way to provide complex localized dosing <i>in vivo</i>. Here we show that it is possible to take advantage of the structure of certain nanomaterials to control release regimes from a scale of hours to months. Graphene oxide (GO) is a two-dimensional charged nanomaterial that can be used to create barrier layers in multilayer thin films, trapping molecules of interest for controlled release. Protein-loaded polyelectrolyte multilayer films were fabricated using layer-by-layer assembly incorporating a hydrolytically degradable cationic poly(β-amino ester) (Poly1) with a model protein antigen, ovalbumin (ova), in a bilayer architecture along with positively and negatively functionalized GO capping layers for the degradable protein films. Ova release without the GO layers takes place in less than 1 h but can be tuned to release from 30 to 90 days by varying the number of bilayers of functionalized GO in the multilayer architecture. We demonstrate that proteins can be released in sequence with multi-day gaps between the release of each species by incorporating GO layers between protein loaded layers. <i>In vitro</i> toxicity assays of the individual materials on proliferating hematopoietic stem cells (HSCs) indicated limited cytotoxic effects with HSCs able to survive for the full 10 days of normal culture in the presence of Poly1 and the GO sheets. This approach provides a new route for storage of therapeutics in a solid-state thin film for subsequent delivery in a time-controlled and sequential fashion