Additional file 1: Figure S1. of Multiple genetically engineered humanized microenvironments in a single mouse

Selection and conformation of lentivial transfected mouse stromal cells. (A) Flow cytometric analysis of GFP mBMSC, (B) Culture-expanded genetically engineered mBMSCs. (Scale bar, 200μm). Figure S2. Characterized secretion of human cytokines from genetically engineered stromal cells in 1 and 3 weeks in vitro culture. Figure S3. hSDF1a ELISA in mouse blood serum. Control mice without scaffold implantation showed a background level of SDF1a signal due to cross-reactivity. This level was used as a baseline and was also observed in growth arrested, which was concluded, as undetectable. The other groups showed measurable levels above background and were concluded to be true hSDF-1a detection. Figure S4. SEM images of growth-competent genetically engineered stromal cell-seeded scaffolds. (A) Cross-sectional images of human soluble factor secreting engineered stromal cell-seeded scaffolds after 6 weeks subcutaneous implantation. Except hTNFa, entire pores were completely filled with tissue cells with no hematopoietic components. (B) Closed-up image of growing engineered stromal cell-seeded scaffolds. Figure S5. Examples of semi-quantitative image analysis using ImageJ. (A) Collagen fiber area estimation from a Masson’s Trichrome staining image, (B) Vasculature area estimation from an immunohistostaining mCD31 and DAPI image. Figure S6. Long-term maintenance of inflammation-mimicking tissue microenvironment indirectly indicates survival and function of growth-arrested hTNFa secreting engineered stromal cells in the implanted scaffolds. (DOCX 2962 kb

Capture and Printing of Fixed Stromal Cell Membranes for Bioactive Display on PDMS Surfaces

Poly­(dimethylsiloxane) (PDMS) has emerged as an extremely useful polymer for various biological applications. The conjugation of PDMS with bioactive molecules to create functional surfaces is feasible yet limited to a single-molecule display with imprecise localization of the molecules on PDMS. Here we report a robust technique that can transfer and print the membrane surface of glutaraldehyde-fixed stromal cells intact onto a PDMS substrate using an intermediate polyvinylalcohol (PVA) film as a transporter system. The cell-PVA film capturing the entirety of surface molecules can be peeled off and subsequently printed onto PDMS while maintaining the spatial display of the original cell surface molecules. Proof-of-concept studies are described using human bone marrow stromal cell membranes including a demonstration of the bioactivity of transferred membranes to capture and adhere hematopoietic cells. The presented process is applicable to virtually any adherent cell and can broaden the functional display of biomolecules on PDMS for biotechnology applications

Alteration in leukocyte migration after MSC-CM treatment.

<p>(A) Experimental design of adoptive transfer study. Gal-N injured rats were treated with vehicle or MSC-CM followed by infusion of In¹¹¹-labeled leukocytes. SPECT images were acquired at t = 0, 3, and 24 hr. for MSC-CM (B–D) and vehicle (E–G) treated rats, respectively.</p

MSC-CM reverses FHF in a cell mass-dependent manner.

<p>(A) Kaplan-Meier survival analysis of Gal-N administered rats treated with concentrated MSC-CM. (B) Dose response of animal survival 72 hours after liver failure induction as a function of MSC mass from which MSC-CM was derived. Controls received vehicle or fibroblast conditioned medium (fibroblast-CM). Time points of interventions are stated above survival plots. Results for both panels are cumulative data of two independent experiments using different batches of MSC-CM (N = 8 per each group). <i>P</i>-value determined by Log Rank Test.</p

MSC-CM treatment inhibits immune cell infiltration and hepatobiliary cell change in Gal-N injured liver tissue.

<p>Representative H&E histology sections of liver tissue from Gal-N injured rats 36 hours post-treatment with vehicle (A,C) or MSC-CM (B, D). Scale bars are indicated on the micrographs. Images (A, B) and (C,D) are captured 10× and 20× magnification, respectively.</p

Infusion of MSC lysate provides a survival trend in an animal model of FHF.

<p>Sprague-Dawley rats were administered lethal intraperitoneal injections of a hepatotoxin. Animals were treated with i.v. injections of MSCs, or MSC lysates from the same cell mass (2×10⁶ cells). Controls received vehicle or NIH 3T3-J2 fibroblast components. Kaplan-Meier survival analysis of Gal-N administered rats treated with cell transplants or lysates. Time points of interventions are stated above survival plots. Results are cumulative data of two independent experiments (N = 8 per each group) using different batches of MSCs. <i>P</i>-value determined by Log Rank Test.</p

MSC-CM is composed of high levels of chemokines that correlate with survival benefit seen in FHF.

<p>Serum-free MSC-CM was analyzed using an antibody array for 174 specified proteins. (A) Densiometry of spotted antibody array results. Data are presented as spot intensity relative to the negative control and normalized to positive control. (B) Pie chart showing cluster analysis of MSC secreted proteins based on reported function. MSC-CM was fractionated over a heparin-agarose column into heparin bound and unbound fractions. (C) Kaplan-Meier survival analysis of Gal-N administered rats treated with the (+) heparin MSC-CM and (−) heparin MSC-CM. Time points of interventions are stated above survival plots. Results for (C) are cumulative data of two independent experiments using different batches of MSC-CM (N = 8 per each group). <i>P</i>-value determined by Log Rank Test.</p

Enhanced delivery of IL-6 by MSC transplants compared to MSC conditioned medium.

<p>(A) Serum profiles of human IL-6 after IV administration of concentrated conditioned medium into C57Bl/6 mice. The plot was normalized to the dose of conditioned medium that was contributed by 1×10⁶ cells. Pharmacokinetic parameters (B) Cmax, (C) AUC, (D) Tmax, (E) Half-life, and (F) Elimination constant were calculated for IL-6 exposure by cell transplants compared to CM administration. Significant differences between cell transplants compared to CM whereby higher levels of IL-6 and longer artificial duration was observed in plasma after cell transplantation. Time points for serum analyses were 0.5, 8, and 24 hours after cell or media injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P>0.01.</p

Combined pharmacokinetic monitoring of MSCs and MSC-secreted IL-6.

<p>(A) Bioluminescent images of C57Bl/6 mice over a period of three days after IV cell administration of one million luciferase-engineered human MSCs. (B) Photon flux of bioluminescent signal over time after IV cell administration. Durable BLI signals were detected up to 24 hours in mice that were injected IV with MSCs. (C) Serum ELISA measurements of human IL-6 released by IV cell transplants over time. Time points for serum and imaging analyses were 0.5, 8, 24, and 72 hours after cell injection. Pooled mouse serum was serially analyzed as batches of N = 5.</p

Golgi-dependent secretion mechanism of MSC-derived IL-6 in vivo.

<p>Brefeldin A pre-treatment of MSCs was used to evaluate blockade of IL-6 release in vitro and in vivo. (A) MTT assay of MSCs treated at different concentrations of brefeldin. A non-toxic dose of 5 ug/ml was used for functional studies. (B) Human IL-6 levels in vitro after brefeldin pre-treatment. Significant reduction in 24 hour release of IL-6 was observed across all doses. (C) Alteration in serum IL-6 delivery by MSCs pretreated with a Golgi-apparatus inhibitor, Brefeldin A. MSCs were incubated with 5 µg/ml of BFA for one day and then injected into C57Bl/6 mice and compared to untreated MSCs in terms of serum IL-6 delivery. Brefeldin treatment of MSCs led to diminished release of human IL-6 in vitro and in vivo. (D) Area-under-curve analysis of human IL-6 after MSC pre-treatment with brefeldin A and transplantation. Exposure to IL-6 was significantly reduced by inhibition of the Golgi apparatus. Time points for serum analyses were 0.5, 8, and 24 hours after cell injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P>0.01.</p