OUTLINE – TIAN Plagarism Plos One Adipocyte

ABSTRACT
 Increased interest in clinical use of adipose-derived stem cells (ASCs)
 Difficult to deliver cells efficiently due to low # of homing factors
 Human ASCs do not have E-selectin (the directing ligand), but express other things
o Instead has CD44 glycoform
 They converted the CD44 into HCELL (which lets E-selectin work more effectively)
o Did not have negative effects on the cell
 Transplanted these new cells into mice to test
 Conclusion: this might be a simple method to enhance homing of stem cell to bone marrow

INTRODUCTION
 Discussion of stem-cell based treatment
o ES (embryonic stem cell) and iPSC (induced pluripotent stem cell) – ethical and technical
issues with using
o MSC (mesenchymal stem cell) – much easier to use, found in bone marrow
o ASC (adipose-derived stem cell) – found in adipose tissue (fat)
 These are excellent for restorative therapy
 Idea of stem-cell migration
o ASC homing is inefficient because they lose “key homing ligands” during the culture
(growth) process
 Discussion of cell migration in general
o A process that involves shear-resistant adhesion interactions between circular cells and
the endolithium (inside the walls of blood vessels/transportation system)
o Determined by selectins
 Step 1: cell-tethering and rolling
 Step 2: activation of integrins
 Step 3: adhesion by integrins
 Step 4: extravasation
 Discussion of Selectins
o Selectins bind to “carbohydrate determinants” expressed on ligands
o In humans and mice, E-selectin is permanently expressed on the skin and bone marrow
 Discussion of HCELL
o HCELL is the most powerful E- and L-selectin ligand which increases ligand activity
o HCELL is a glycoform of CD44 which can be chemically transformed
o Therefore, the chemical which converts CD44 into HCELL on ASC could help generate
HCELL and increase binding activity
o ** “the proof for this hypothesis” (bad word usage)
 This study tests whether the “enforced fucosylation” (CD44HCELL) on human ASCs improve
migration

MATERIALS AND METHODS

 Obtained ASC from five healthy women
o Procedures were approved by the ethics board
o Consent obtained
 Discussion of how they processed and separated the ASCs (hASC = human ASC)
o At the end of incubation, the differences were detected by red oil staining
 Obtained umbilical cords from three healthy delivery women
o Procedures were approved by the ethics board
 o Consent obtained
 Discussion of how they processed and separated the HUVECs (human umbilical vein endolethial
cells)
 Treatment of the hASCs with chemicals
 Flow Cytometry
o Cells were incubated with “primary monoclonal antibodies” for flow cytometry
o Controls were used
 Cell Proliferation Assay
 Western Blot Analysis
o Gel electrophoresis was performed on the HCELL+ ASCs
 Cell Adhesion Assay
o HUVEC was treated to cause E-selection to be induced
o Performed a static adhesion assay to see how many cells had e-selectin binding after an
hour
 Parallel Plate Flow Chamber Adhesion Assay
o hASCs were tested for adhesion in a parallel plate flow chamber (to simulate blood flow)
 Short Term Homing Assay
o Cells were injected into mice
o HCELL+ hASCs, untreated hASCs, or FTV-sialidase-hASCs were injected into the mice
and dissected 16 hours later to see the rates of adhesion and binding
 In Vivo Bone Formation Studies
o Mice injected with hASC, HCELL+ hASCs, or buffer (as control)
o The mice were killed and testing was performed to determine how much binding
occurred
 Immunofluorescence
o Staining of bone sections was performed to test for human osteoid tissue
 Statistical Analysis
o T-testing was used, any p < .05 was considered significant

RESULTS
 Expression of Homing Receptors on hASCs
o hASC expressed CD44 (flow-cytometry to determine this)
o hASC did not express E-selectin
 Generation of HCELL
o After fucosylation, cells expressed strong expression of HCELL
o Does not compromise cell viability
 Static/Dynamic Assays
o Strong HCELL expression induces strong binding on endolethial E-selectin (static)
o HCELL expression produces a “robust rolling response” on endolethial E-selectin
(dynamic)
 Ex Vivo Fucosylatoin
o HCELL+ hASCs accumulated
o Typo: untreat
o HCELL on hASCs increases tendency of the cells to migrate to bone marrow
 Infiltrated HCELL+ hASCs turned into osteoids
o Extravasated cells can lodge within marrow and generate human osteoid in mouse bone
marrow

DISCUSSION
 ASCs are easily harvested and have no ethical issues
 Use an alpha-1,3-fucosyltransferaase (FTV) to engineer hASCs
 Using adhesion and flow chamber assays, results show that expression of HCELL on hASCs
increase E-selection binding
 HCELL expression was NOT LONG LASTING (only lasted about 24 hours)
 Not likely to interfere with the long term function of ASC
 “proof of principle”