Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells

The anterior cruciate ligament (ACL) usually fails to heal after rupture mainly due to the inability of the cells within the ACL tissue to establish an adequate healing process, making graft reconstruction surgery a necessity. However, some reports have shown that there is a healing potential of ACL with primary suture repair. Although some reports showed the existence of mesenchymal stem cell-like cells in human ACL tissues, their origin still remains unclear. Recently, blood vessels have been reported to represent a rich supply of stem/progenitor cells with a characteristic expression of CD34 and CD146. In this study, we attempted to validate the hypothesis that CD34- and CD146-expressing vascular cells exist in hACL tissues, have a potential for multi-lineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured ACL. Immunohistochemistry and flow cytometry analysis of hACL tissues demonstrated that it contains significantly more CD34 and CD146-positive cells in the ACL ruptured site compared with the noninjured midsubstance. CD34+CD45- cells isolated from ACL ruptured site showed higher expansionary potentials than CD146+CD45- and CD34-CD146-CD45- cells, and displayed higher differentiation potentials into osteogenic, adipogenic, and angiogenic lineages than the other cell populations. Immunohistochemistry of fetal and adult hACL tissues demonstrated a higher number of CD34 and CD146-positive cells in the ACL septum region compared with the midsubstance. In conclusion, our findings suggest that the ACL septum region contains a population of vascular-derived stem cells that may contribute to ligament regeneration and repair at the site of rupture. © 2012 Mary Ann Liebert, Inc

Inkjet-based biopatterning of bone morphogenetic protein-2 to spatially control calvarial bone formation

The purpose of this study was to demonstrate spatial control of osteoblast differentiation in vitro and bone formation in vivo using inkjet bioprinting technology and to create three-dimensional persistent bio-ink patterns of bone morphogenetic protein-2 (BMP-2) and its modifiers immobilized within microporous scaffolds. Semicircular patterns of BMP-2 were printed within circular DermaMatrix™ human allograft scaffold constructs. The contralateral halves of the constructs were unprinted or printed with BMP-2 modifiers, including the BMP-2 inhibitor, noggin. Printed bio-ink pattern retention was validated using fluorescent or 125I-labeled bio-inks. Mouse C2C12 progenitor cells cultured on patterned constructs differentiated in a dose-dependent fashion toward an osteoblastic fate in register to BMP-2 patterns. The fidelity of spatial restriction of osteoblastic differentiation at the boundary between neighboring BMP-2 and noggin patterns improved in comparison with patterns without noggin. Acellular DermaMatrix constructs similarly patterned with BMP-2 and noggin were then implanted into a mouse calvarial defect model. Patterns of bone formation in vivo were comparable with patterned responses of osteoblastic differentiation in vitro. These results demonstrate that three-dimensional biopatterning of a growth factor and growth factor modifier within a construct can direct cell differentiation in vitro and tissue formation in vivo in register to printed patterns. © 2010 Mary Ann Liebert, Inc

Platelet-Rich Plasma Promotes the Proliferation of Human Muscle Derived Progenitor Cells and Maintains Their Stemness

Human muscle-derived progenitor cells (hMDPCs) offer great promise for muscle cell-based regenerative medicine; however, prolonged ex-vivo expansion using animal sera is necessary to acquire sufficient cells for transplantation. Due to the risks associated with the use of animal sera, the development of a strategy for the ex vivo expansion of hMDPCs is required. The purpose of this study was to investigate the efficacy of using platelet-rich plasma (PRP) for the ex-vivo expansion of hMDPCs. Pre-plated MDPCs, myoendothelial cells, and pericytes are three populations of hMDPCs that we isolated by the modified pre-plate technique and Fluorescence Activated Cell Sorting (FACS), respectively. Pooled allogeneic human PRP was obtained from a local blood bank, and the effect that thrombin-activated PRP-releasate supplemented media had on the ex-vivo expansion of the hMDPCs was tested against FBS supplemented media, both in vitro and in vivo. PRP significantly enhanced short and long-term cell proliferation, with or without FBS supplementation. Antibody-neutralization of PDGF significantly blocked the mitogenic/proliferative effects that PRP had on the hMDPCs. A more stable and sustained expression of markers associated with stemness, and a decreased expression of lineage specific markers was observed in the PRP-expanded cells when compared with the FBS-expanded cells. The in vitro osteogenic, chondrogenic, and myogenic differentiation capacities of the hMDPCs were not altered when expanded in media supplemented with PRP. All populations of hMDPCs that were expanded in PRP supplemented media retained their ability to regenerate myofibers in vivo. Our data demonstrated that PRP promoted the proliferation and maintained the multi-differentiation capacities of the hMDPCs during ex-vivo expansion by maintaining the cells in an undifferentiated state. Moreover, PDGF appears to be a key contributing factor to the beneficial effect that PRP has on the proliferation of hMDPCs. © 2013 Li et al

Stem Cells in Drug Screening for Neurodegenerative Disease

Because the average human life span has recently increased, the number of patients who are diagnosed with neurodegenerative diseases has escalated. Recent advances in stem cell research have given us access to unlimited numbers of multi-potent or pluripotent cells for screening for new drugs for neurodegenerative diseases. Neural stem cells (NSCs) are a good model with which to screen effective drugs that increase neurogenesis. Recent technologies for human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) can provide human cells that harbour specific neurodegenerative disease. This article discusses the use of NSCs, ESCs and iPSCs for neurodegenerative drug screening and toxicity evaluation. In addition, we introduce drugs or natural products that are recently identified to affect the stem cell fate to generate neurons or glia

Isolation of muscle-derived stem/progenitor cells based on adhesion characteristics to collagen-coated surfaces

Our lab developed and optimized a method, known as the modified pre-plate technique, to isolate stem/ progenitor cells from skeletal muscle. This method separates different populations of myogenic cells based on their propensity to adhere to a collagen I-coated surface. Based on their surface markers and stem-like properties, including self-renewal, multi-lineage differentiation, and ability to promote tissue regeneration, the last cell fraction or slowest to adhere to the collagen-coated surface (pre-plate 6; pp6) appears to be early, quiescent progenitor cells termed muscle-derived stem/progenitor cells (MDSPCs). The cell fractions preceding pp6 (pp1-5) are likely populations of more committed (differentiated) cells, includingfibroblast- and myoblast-like cells. This technique may be used to isolate MDSPCs from skeletal muscle of humans or mice regardless of age, sex or disease state, although the yield of MDSPCs varies with age and health. MDSPCs can be used for regeneration of a variety of tissues including bone, articular cartilage, skeletal and cardiac muscle, and nerve. MDSPCs are currently being tested in clinical trials for treatment of urinary incontinence and myocardial infarction. MDSPCs from young mice have also been demonstrated to extend life span and healthspan in mouse models of accelerated aging through an apparent paracrine/ endocrine mechanism. Here we detail methods for isolation and characterization of MDSPCs. © Springer Science+Business Media, LLC 2013

Effect of Solution Composition of Plasmid DNA on Gene Transfection Following Liver Surface Administration in Mice

We investigated the effect of plasmid DNA (pDNA) solution composition on gene transfection following liver surface administration in mice. Gene transfection experiments in situ and in vivo were performed using the following pDNA solutions: dextrose solution, NaCl solution, phosphate buffer, phosphate-buffered saline, Tris/HCl buffer with EDTA, Tris/HCl buffer with EDTA and Triton X-100, and water. In in situ experiments, we used a glass cylindrical diffusion cell that limited the contact area between the liver surface and the naked pDNA solution. The gene transfection at the site of diffusion cell attachment increased in hypotonic solution, and decreased in hypertonic solution, compared with isotonic solution. In in vivo experiments, instillation of naked pDNA solution onto the liver surface using a micropipette caused no significant differences in gene transfection in the applied lobe. These results suggest that it is important to select the optimal pDNA solution composition to control the gene transfection