This month marks the release of JoVE's 100th issue, and we now have 10 scientific sections: Biology, Neuroscience, Immunology & Infection, Medicine, Bioengineering, Engineering, Chemistry, Behavior, Environment, and Developmental Biology.
In the field of Developmental Biology, sometimes you'll come across the term "buffy coat"-which might sound like a pet's coat color, or maybe a manicure, or maybe the leather under-armor worn by 17th Century cavalry. But a buffy coat is a product of centrifuging whole blood, and refers to the cloudy layer containing white blood cells, which forms between the red blood cells and the plasma.
Meraviglia et al. collect the buffy coats and turn peripheral blood mononuclear cells (PBMNCs) into induced pluripotent stem cells (iPSCs). Instead of inducing pluripotency as usual with viral vectors, our authors reprogram the cells using non-integrating plasmids. The resulting iPSC colonies show normal morphology, stable karyotypes, and significant expression of pluripotency markers. As far as we know, this is the first report of deriving virus-free iPSCs from frozen buffy coats, which are commonly stored in biobanks, and could become a valuable source of iPSCs for regenerative medicine.
In JoVE Bioengineering, we know that cells can behave differently depending on the stiffness of the extracellular matrix, which is measured in kiloPascals (kPA). Metastatic cancer cells, in particular, show higher traction stress than non-metastatic cells, so scientists examine tumor cell invasion and metastasis using traction force assays. However, traction data are lacking for primary tumor cells. Therefore, Yakut Ali et al. extract primary cells from human tumors and culture them on hydrogel substrates for traction cytometry. This technique may lead to the use of biophysical parameters, such as tumor cell stiffness and traction, in cancer prognostics.
In JoVE Behavior, we feature protocols that deal with cognition and learning in children. In the experimental paradigm by Sargent et al., a mobile hangs above an infant, and a light-emitting diode (LED) is secured to the infant's leg. When it reaches a certain height, the LED activates the mobile. The infants discover that the mobile will spin and play music if they kick their legs up. A motion capture system records these movements so the learning process can be quantified.
Also in JoVE Behavior, children with speech impairments may benefit from a speech-generating device (SGD), which produces an electronic voice using a synthesizer. Some devices have dynamic interfaces, so users can access new words by changing levels. Cognitive factors can affect the ability to navigate through these levels, so Robillard et al. present a detailed protocol for assessing how cognitive factors contribute to navigational abilities. This could, in turn, guide clinicians to select the appropriate speech-generating device for a patient.
Manon Robillard1, Chantal Mayer-Crittenden1, Annie Roy-Charland2, Michèle Minor-Corriveau1, Roxanne Bélanger1
1Speech-Language Pathology Program, Laurentian University, 2Psychology Department, Laurentian University
Children who have complex communication needs can benefit from the use of a speech-generating device (SGD). Cognitive skills were identified as having an impact on the ability to navigate between levels of SGDs. This protocol describes the steps needed for Speech-language Pathologists to assess cognitive skills and navigational abilities.
Barbara Sargent1, Hendrik Reimann2, Masayoshi Kubo3, Linda Fetters1
1Division of Biokinesiology & Physical Therapy at the Herman Ostrow School of Dentistry, University of Southern California, 2Department of Kinesiology, Temple University, 3Department of Physical Therapy, Niigata University of Health and Welfare
A method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process.
Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry
M. Yakut Ali1, Sandeep V. Anand1, Krishnarao Tangella2,3, Davendra Ramkumar2,3, Taher A. Saif1,4
1Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, 2College of Medicine, University of Illinois at Urbana-Champaign, 3Provena Covenant Medical Centre, 4Micro and Nanotechnology Laboratory, University of Illinois at Urbana-Champaign
A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.
Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids
Viviana Meraviglia*1, Alessandra Zanon*1, Alexandros A. Lavdas1, Christine Schwienbacher1, Rosamaria Silipigni2, Marina Di Segni2, Huei-Sheng Vincent Chen3, Peter P. Pramstaller1, Andrew A. Hicks1, Alessandra Rossini1
1Center for Biomedicine, European Academy Bozen/Bolzano (EURAC), 2Laboratory of Medical Genetics, Fondazione IRCCS Ca´ Granda, Ospedale Maggiore Policlinico, 3Del E. Webb Center for Neuroscience, Aging & Stem Cell Research, Sanford-Burnham Medical Research Institute
Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.
No conflicts of interest declared.