Lab Animal Models and Scientific Approach
Supinder Bedi, PhD
Overview: The focus of the laboratory is to define the neuroinflammatory response to traumatic brain injury in terms of microglial polarization. Cellular therapies have been shown to alter the microglial/macrophage phenotype after injury, and we seek to translate those findings into protocols that can be moved into clinical practice.
We are using the cortical contusion injury model to study the therapeutic effects of stem cells after traumatic brain injury (TBI) in a rodent model. We utilize short and long-term assays to study the effectiveness of stem cell therapy.
Cell lines and primary cells:
Multipotent Adult Progenitor Cells (MAPC, Athersys, Inc.)
Human Placental Derived Stem Cells (HPDSC, Celgene)
Autologous bone marrow derived mononuclear cells
Human Amniotic fluid derived mesenchymal stromal/stem cells
Human bone marrow derived mesenchymal stromal/stem cells
Commonly Used Techniques:
Blood-Brain Barrier (BBB) permeability measurements: We have used traditional dye extraction techniques with spectrophotometric detection of Evan’s Blue dye in solution for this short-term (72h) outcome measure, but recently published our new method using an imaging approach (J Surg Res. 2014 Aug;190(2):628-33).
Morris Water Maze: The MWM is used to assess long-term spatio-temporal memory as well as learning paradigms. Assessments are performed at 120 days post injury to ascertain true long-term effects of cell therapies.
DT-MRI- 9T DT-MRI imaging is performed in a serial fashion to ascertain the effects of cell therapy on WM tracts and volumetrics as determined by FA, MD and RD measurements in regions of interest after TBI.
Immunohistochemistry and microglial quantification: Microglia are characterized by both morphology and cell surface markers in tissue sections. Alternatively, we have developed a flow cytometry based method to quantify microglial populations. These complimentary techniques are used to define the neuroinflammatory response to injury and are a critical read-out for cell therapy efficacy. Use of TSPO immunohistochemistry is useful as a proof of concept for translating this marker of microglial activation to PET-CT/MRI imaging in pre-clinical and clinical applications.
Scott Olson, PhD
Overview: The focus of the laboratory is to study the neuroinflammatory reflex arc that occurs after TBI, and mechanisms of interrupting the amplification of the inflammatory response using cell based therapeutics.
We study TBI in rodents created using a controlled cortical impact. Briefly, we impact the surface of the dura with an electromagnetically driven piston such that we can precisely control the speed of the piston, the depth of penetration, and the amount of time it remains in place, as well as changing the size of the piston itself. This creates a reliable, survivable, and replicable injury unilaterally to the cortex and is a technique that is widely adopted in TBI research.
We don’t currently use any genetically modified animals.
Cell lines and primary cells:
We have cryopreserved bone marrow mononuclear cell fractions and isolated bone marrow derived mesenchymal stem/stromal cells (MSC) from approximately 12 commercially acquired bone marrow aspirates, 3 enriched bone marrow aspirates from patients enrolled in our clinical trials, and 7 pediatric organ donors. We have isolated adipose-derived MSC from 3 organ donors. We have isolated and cryopreserved umbilical cord blood from approximately 10 cords. We have isolated pig bone marrow MSC. We use THP-1, U937, and RAW264.7 monocytic cell lines and Jurkat T-cell line.
Commonly used assays and techniques:
Our group regularly uses ELISA to quantify cytokines, proteins, and metabolites, western blot, In-cell western, immunofluorescence, immunohistochemistry of brain slices, quantitative image analysis, vascular permeability assays (for blood-brain barrier), tissue water analysis (for edema), kinetic assays to quantify acetylcholine, viability, flow cytometry, adherence assays, alginate encapsulation of live cells, qPCR, differentiation, and proliferation assays.
Fabio Triolo, PhD
Overview: Despite dramatic advancements in medical and surgical care, effective clinical therapies for neurological injury are limited. The past decade’s rapid advancement in stem cell biology and neurology has generated a growing body of literature supporting the use of various progenitor cell types to treat acute neurological injuries. In this context, my lab is actively involved in several research endeavors of the Program of Regenerative Medicine. These include the development of human adult and fetal cell-based therapies to improve neurological conditions, such as anoxic brain injury at birth, cerebral palsy, traumatic brain injury and stroke, all of which are still unmet medical needs that have not been able to be satisfied by conventional healthcare therapies. My research interests also include the development of innovative autologous tissue engineering applications based on extra-embryonic tissues (e.g., Amniotic Fluid, Wharton’s Jelly).
Cleft lip and cleft palate (CLP) represent nearly 1/3 of all birth defects. Autologous bone grafts are the gold standard treatment, but are invasive and have potentially serious complications that lead to the need of additional surgeries. Another strategy based on using biomaterials seeded with bone marrow (BM) stem cells has proven promising, but BM harvest is too invasive to use in CLP repair in newborns, thus alternative strategies are needed. We are using a 7 x 4 x 3 mm critical-size alveolar bone defect model in the rat to investigate the use of native Wharton’s Jelly (WJ) in the repair of CLP. WJ, the connective tissue matrix of the umbilical cord, is a gelatinous substance comprised of proteoglycans and various isoforms of collagen and is rich in perinatal stem cells. It is a natural “tissue engineering” construct that provides a scaffold derived from the recipient’s own molecules, naturally seeded with the recipient’s own stem cells, and is thus immunologically inert. Since WJ is typically discarded as post-delivery medical waste, its use does not pose ethical concerns and its harvest is completely non-invasive. Our hypothesis is that inclusion of WJ in the alveolar pocket of CLP patients at the time of palate repair will enhance bone growth and accelerate healing, proving to be an ideal adjunct to orofacial cleft repair.The success of this approach would represent a paradigm shift in the treatment of CLP patients, significantly anticipating the timing of surgical correction and reducing or eliminating the need for subsequent bone grafting.
Clinical-grade human amniotic fluid derived mesenchymal stromal cells isolated in my lab are also being used in a rat model of congenital diaphragmatic hernia within a project aimed at developing a tissue engineered diaphragm replacement, led by Dr. Yong Li.
Cell lines and primary cells:
- Clinical-grade human mesenchymal stromal/stem cells derived from:
- Amniotic fluid of normal gestations
- Amniotic fluid of patients undergoing amnioreduction during treatment of Twin-to-Twin Transfusion syndrome
- Amniotic fluid of patients undergoing prenatal repair of myelomeningocele
- Bone marrow
- Wharton’s Jelly
- Human mononuclear cells derived from bone marrow and cord blood
Commonly used techniques:
- Clinical-grade human tissue processing, stem cell production, cryopreservation and tissue vitrification in compliance with current Good Manufacturing Practice (cGMP) of the FDA
- Biophysical characterization of human native Wharton’s Jelly (WJ) using atomic force microscopy and shear rheology, flat panel CT imaging on living animals to determine the effect of WJ implant on bone regrowth and healing in time in an alveolar defect model representative of cleft palate surgery
Pam Wenzel, PhD
Overview: Shear stress, or frictional force, modulates the behavior of mesenchymal stem cells, and impacts proliferation, cell survival, and fate decisions. A growing body of literature suggests that these types of cells can suppress unchecked inflammatory signaling and innate immune response in patients. Consequently, a primary area of interest is to determine how mechanical force alters the biology of mesenchymal stem cells, including their ability to modulate anti-inflammatory programs and vascular permeability. We utilize culture-based assays, cellular phenotyping, and mesenchymal stem cell-based therapy models of traumatic brain injury in the rat as readouts of response to mechanical stimuli. We are comparing the immunomodulatory potential of mesenchymal stem cells from various tissue sources by in vitro inflammatory cytokine assays and transplantation into rats exposed to controlled cortical impact to induce head trauma.
The objective of the research sponsored by our NIH and ASH grants is to identify biomechanically induced pathways that promote hematopoietic fate during embryonic development. These studies rely on the use of mice to model human development and aim to address three critical areas of interest: 1) identification of the cell types that respond to physical force by differentiation into blood lineages, 2) interrogation of pathways important for blood development that are regulated by force, and 3) evaluation of soluble agents/drugs that can stimulate a signature of pathways similar to that activated by force. For this work, we utilize several mouse strains, including transgenic, knockout, and inbred strains of mice, which collectively provide a means of monitoring gene activity with fluorescence and enhancing or ablating gene function.
Animal protocols: HSC-AWC-12-011, HSC-AWC-14-015
Human IRB protocols: HSC-MS-11-0593, HSC-MS-0560
Biosafety protocol: IBC-14-050
Mice – (all are genetically modified, except first 3 inbred strains)
- B6.SJL-Ptprca Pep3b/BoyJ (referred to as B6.SJL)
- F1 Ly5.1/Ly5.2 (referred to as CD45.1/.2)
- NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (also called NSG)
- Rag2tm1Fwa II2rgtm1Wjl (also called Rag2 Il2rg double knockout or Rag2γc)
- B6.SJL.Rag2tm1Fwa II2rgtm1Wjl (referred to as Rag2γc CD45.1)
- F1 Rag2γc Ly5.1/Ly5.2 (Rag2γc CD45.1/.2)
- C57BL/6-Tg(UBC-GFP)30Scha/J (referred to as UBC-GFP)
- B6.Cg-Tg(ACTB-mRFP1)1F1Hadj/J (referred to as ACT-RFP)
- Gt(ROSA)26Sortm1(EYFP)Cos (referred to as Rosa26R-EYFP)
- Notch1tm3(cre)Rko (referred to as NIP-Cre)
- Gt(Rosa)26Sortm1(cre/ERT2)Tyj (tamoxifen-inducible cre; referred to as ERT2-Cre)
- Psen1tm1Vln (referred to as Psen1)
- ERT2-Cre; Psen1 double homozygous
- B6.Cg-Tg(Ly6a-EGFP)G5Dzk/J (referred to as Ly6a-EGFP)
- B6.Ncx1 (sodium channel knockout; referred to as Ncx1)
Cell lines and primary cells:
- Cells (animal) Mouse cell lines NIH-3T3, ES cells (e.g. AINV15, KH2), MEFs (e.g. CF1 gamma irradiated feeders), OP9 bone marrow stromal cells
- Cells (animal) Mouse embryonic hematopoietic tissues
- Cells (human) HUVEC
- Cells (human) Human Vascular Smooth Muscle Cells (VSMC)
- Cells (human) Human bone marrow mesenchymal stem cells
- Cells (human) Human adipose-derived mesenchymal stem cells
- Cells (human) Human amniotic fluid mesenchymal stem cells
Commonly used techniques:
Using custom fluidics platforms, we are modulating the shear stress present in the cellular environment and evaluating its impact on cellular potential by a number of biochemical, genetic, and functional approaches. Phenotypic and functional measurements are made by flow cytometry, colony formation assays, biochemical quantification of components in mechanotransductive signaling (ELISA, immunoblotting, and Griess assays), and transplantation into adult recipient mice or rats. Metalloprotease activity is measured by chemiluminescence, motility by time lapse imaging, and activation of genetic signaling pathways by qRT PCR or global gene expression profiling, confocal immunofluorescent staining, and immunoblotting.