Improved Islet Yields From Macaca Nemestrina and Marginal Human Pancreata After Two-Layer Method Preservation and Endogenous Trypsin Inhibition

We tested whether two-layer method (TLM) pancreas preservation and trypsin inhibition (Pefabloc) during processing allows longer preservation while retaining or improving viable islet recovery. Non-marginal primate (Macaca nemestrina) and marginal human (ischemic or preservation-injured) pancreata were processed with a research-oriented pan technique (Seattle method). Organs were processed upon arrival (± Pefabloc), or after TLM or University of Wisconsin solution (UW) preservation (+ Pefabloc). Islet yield, viability, and function were assessed. Pefabloc increased M. nemestrina islet yields from 9696 ± 1749 IE/g to 15 822 ± 1332 IE/g (p \u3c 0.01). Two-layer method preservation (\u3c 6 h) further increased yields, to 23 769 ± 2773 IE/g (vs. + Pefabloc; p \u3c 0.01). Similarly, Pefabloc increased marginal human islet yields from 2473 ± 472 IE/g to 4723 ± 1006 IE/g (p \u3c 0.04). This increase was maintained after lengthy TLM preservation (\u3e 30 h; 4801 ± 1066 IE/g). We also tested the applicability of TLM preservation (23.5 ± 3.2 h) to the processing of marginal human pancreata by the Edmonton/Immune Tolerance Network clinical protocol. Islet yield and function approached published results of pancreata processed 4.8 ± 0.8 h after organ recovery (p = 0.06). Pefabloc, and TLM vs. UW preservation, prolonged the tolerable interval between organ recovery and islet isolation. Islet yield, viability, and functionality improved from both marginal and nonmarginal pancreata

Improved Islet Yields From Macaca Nemestrina and Marginal Human Pancreata After Two-Layer Method Preservation and Endogenous Trypsin Inhibition

We tested whether two-layer method (TLM) pancreas preservation and trypsin inhibition (Pefabloc) during processing allows longer preservation while retaining or improving viable islet recovery. Non-marginal primate (Macaca nemestrina) and marginal human (ischemic or preservation-injured) pancreata were processed with a research-oriented pan technique (Seattle method). Organs were processed upon arrival (± Pefabloc), or after TLM or University of Wisconsin solution (UW) preservation (+ Pefabloc). Islet yield, viability, and function were assessed. Pefabloc increased M. nemestrina islet yields from 9696 ± 1749 IE/g to 15 822 ± 1332 IE/g (p \u3c 0.01). Two-layer method preservation (\u3c 6 h) further increased yields, to 23 769 ± 2773 IE/g (vs. + Pefabloc; p \u3c 0.01). Similarly, Pefabloc increased marginal human islet yields from 2473 ± 472 IE/g to 4723 ± 1006 IE/g (p \u3c 0.04). This increase was maintained after lengthy TLM preservation (\u3e 30 h; 4801 ± 1066 IE/g). We also tested the applicability of TLM preservation (23.5 ± 3.2 h) to the processing of marginal human pancreata by the Edmonton/Immune Tolerance Network clinical protocol. Islet yield and function approached published results of pancreata processed 4.8 ± 0.8 h after organ recovery (p = 0.06). Pefabloc, and TLM vs. UW preservation, prolonged the tolerable interval between organ recovery and islet isolation. Islet yield, viability, and functionality improved from both marginal and nonmarginal pancreata

Cell-Free Amniotic Fluid and Regenerative Medicine: Current Applications and Future Opportunities

Amniotic fluid (AF) provides critical biological and physical support for the developing fetus. While AF is an excellent source of progenitor cells with regenerative properties, recent investigations indicate that cell-free AF (cfAF), which consists of its soluble components and extracellular vesicles, can also stimulate regenerative and reparative activities. This review summarizes published fundamental, translational, and clinical investigations into the biological activity and potential use of cfAF as a therapeutic agent. Recurring themes emerge from these studies, which indicate that cfAF can confer immunomodulatory, anti-inflammatory, and pro-growth characteristics to the target cells/tissue with which they come into contact. Another common observation is that cfAF seems to promote a return of cells/tissue to a homeostatic resting state when applied to a model of cell stress or disease. The precise mechanisms through which these effects are mediated have not been entirely defined, but it is clear that cfAF can safely and effectively treat cutaneous wounds and perhaps orthopedic degenerative conditions. Additional applications are currently being investigated, but require further study to dissect the fundamental mechanisms through which its regenerative effects are mediated. By doing so, rational design can be used to fully unlock its potential in the biotechnology lab and in the clinic

Induction of polyploidization in leukemic cell lines and primary bone marrow by Src kinase inhibitor SU6656

Megakaryocytes (MKs) undergo successive rounds of endomitosis during differentiation, resulting in polyploidy (typically, 16-64N). Previous studies have demonstrated that this occurs through an interruption of normal cell cycle progression during anaphase. However, the molecular mechanism(s) controlling this unique process is undefined. In the present report, we examine the effect of an Src kinase inhibitor, SU6656, on thrombopoietin (TPO)-induced growth and differentiation. Remarkably, when SU6656 (2.5 μM) was added to a megakaryocytic cell line, UT-7/TPO, the cells ceased cell division but continued to accumulate DNA by endomitosis. During this interval, CD41 and CD61 expression on the cell surface increased. Similar effects on polyploidization and MK differentiation were seen with expanded primary MKs, bone marrow from 2 patients with myelodysplastic syndrome, and other cell lines with MK potential. Our data suggest that SU6656 might be useful as a differentiation-inducing agent for MKs and is an important tool for understanding the molecular basis of MK endomitosis

Human mesenchymal stromal cell (MSC) characteristics vary among laboratories when manufactured from the same source material: a report by the Cellular Therapy Team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative

Background: Culture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods and different source materials. The purpose of this study was to assess the impact on MSC characteristics when different laboratories propagated MSCs from cultures initiated with BM aliquots derived from the same donor source material. Methods and Methods: Five aliquots from each of three different BM donors were distributed to five independent laboratories. Three laboratories plated whole BM and two laboratories a mononuclear BM cell fraction. Four laboratories cultured in media supplemented with fetal bovine serum (FBS) and one laboratory used human platelet lysate (hPL). Initial cell seeding densities (i.e., P0) ranged from 19.7 × 103/cm2–282 × 103/cm2 and for second seeding (i.e., P1) 0.05 × 103–5.1 × 103 cells/cm2. Post-thawed MSCs from each laboratory were analyzed for cell viability, immunophenotype, tri-lineage differentiation, fibroblast colony-forming units (CFU-F), gene expression, and immunosuppressive activity. Results: Transit times from BM collection to receipt by laboratories located in the United States ranged from 16.0–30.0 h and from 41.5–71.5 h for a laboratory in Asia. Post-thaw culture derived MSCs rom BM #1, #2, and #3 exhibited viabilities that ranged from 74–92%, 61–96%, and 23–90%, respectively. CFU activity from BM #1, #2, and #3 per 200 MSCs plated averaged 45.1 ± 21.4, 49.3 ± 26.8 and 14.9 ± 13.3, respectively. No substantial differences were observed in immunophenotype, and immunosuppressive activities. Global gene expression profiles of MSCs revealed transcriptome differences due to different inter-laboratory methods and to donor source material with the center effects showing greater molecular differences than source material. Conclusion: Functional and molecular differences exist among MSCs produced by different centers even when the same BM starting material is used to initiate cultures. These results indicated that manufacturing of MSCs by five independent centers contributed more to MSC variability than did the source material of the BM used in this study. Thus, emphasizing the importance of establishing worldwide standards to propagate MSCs for clinical use

Marrow VME for the study of thrombopoiesis <i>in vitro</i>.

<p><b>A</b>. Z-stack projection of confocal fluorescence imaging of megakaryocytes co-cultured within a 3D microvascular system. Green: CD41, red: CD31, blue: nuclei. <b>B</b>. Enlarged view, z-projection of confocal fluorescence images (left panel) and orthogonal views (right two panels) of locations at dotted lines 1 and 2, showing megakaryocytes interacting with the vessel wall (stars) and in the lumen and on the abluminal vessel wall (arrowheads). Green: CD41, red: CD31, blue: nuclei. <b>C</b>. (i) Zoomed view of megakaryocyte indicated in A (arrowhead) showing CD41a+ (green) and nucleus staining (blue) (ii) 3D reconstruction of the nucleus lobes from the megakaryocyte in i. <b>D</b>. A TEM image showing a megakaryocyte with four nucleus lobes close to a vessel. <b>E</b>. Megakaryocyte lobe counts near and far from the vessel wall shows more mature megakaryocytes are located closer to the vessel wall.</p

Microvasculature-directed thrombopoiesis in a 3D <i>in vitro</i> marrow microenvironment

<div><p>Vasculature is an interface between the circulation and the hematopoietic tissue providing the means for hundreds of billions of blood cells to enter the circulation every day in a regulated fashion. The precise mechanisms that control the interactions of hematopoietic cells with the vessel wall are largely undefined. Here, we report on the development of an <i>in vitro</i> 3D human marrow vascular microenvironment (VME) to study hematopoietic trafficking and the release of blood cells, specifically platelets. We show that mature megakaryocytes from aspirated marrow as well as megakaryocytes differentiated in culture from CD34+ cells can be embedded in a collagen matrix containing engineered microvessels to create a thrombopoietic VME. These megakaryocytes continue to mature, penetrate the vessel wall, and release platelets into the vessel lumen. This process can be blocked with the addition of antibodies specific for CXCR4, indicating that CXCR4 is required for megakaryocyte migration, though whether it is sufficient is unclear. The 3D marrow VME system shows considerable potential for mechanistic studies defining the role of marrow vasculature in thrombopoiesis. Through a stepwise addition or removal of individual marrow components, this model provides potential to define key pathways responsible for the release of platelets and other blood cells.</p></div

Characterization of generated platelet-like particles.

<p><b>A</b>. Flow cytometry of whole blood, washed platelets, and collected particles from MK vessels at day five of culture showing granularity (SSC-A) and size (FSC-A). Gating was based on the platelet population of the whole blood sample. <b>B.</b> CD41a and CD42b expression of gated population from whole blood, washed platelets, and collected particles at day five. <b>C-D.</b> Immunofluorescence images of un-activated and thrombin-activated particles stained for CD41a and β-tubulin.</p