Characterization of Mesenchymal Stem Cells from Human Cortical Bone
doi: 10.13052/ijts2246-8765.2016.005
Joseph S. Fernandez-Moure1, Bruna Corradetti2, Trevor Janecek1,
Jeffrey Van Eps1, Matthew Burn1, Bradley K.Weiner1,
Pranela Rameshwar3 and Ennio Tasciotti2
1The Methodist Hospital Research Institute, Department of Surgery,
Houston Methodist Hospital, Houston, Texas, USA
2Houston Methodist Research Institue, Houston, Texas, USA
3Rutgers New Jersey Medical School, Newark, New Jersey, USA
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Abstract:
Background Context: Mesenchymal stem cells (MSC) are being used for
spine and orthopaedic surgical and research applications. Bone marrow and
fat are the most commonly used sources of these cells.
Purpose: To describe a new technique allowing the isolation and expansion
of human MSC from cortical bone.
Study Design: MSC from human cortical bone (vertebral lamina) were
isolated, expanded, and verified in vitro.
Methods: Human MSC were isolated from laminar bone obtained during
surgery (decompression/laminectomy). They were then cultured and assessed
using fluorescence-activated cell sorting techniques forMSCmarkers, colonyforming
unit assays, and multilineage differentiation.
Results: Isolated and cultured cells demonstrated MSC markers and trilineage
differentiation confirming their stemness.
Conclusion: Anovel method for the isolation of MSC from cortical bone has
been described. These cells have significant current and future application
in spine and orthopaedic surgery; and both the source of the cells and
particular characteristics of the cortical bone derived MSC have advantages
over currently used MSC obtained from other sources.
Keywords:
Levels of Stromal Derived Factor-1 (SDF-1) and Brain Derived Neurotropic Factor (BDNF) and Very Small Embryonic-Like Cells (VSEL) in Ischemic Stroke Patients
doi: 10.13052/ijts2246-8765.2016.004
Bayu Winata Putera1,2, Cynthia R. Sartika2, Andi Wijaya1,2,2
Irawan Yusuf1 and Aw Tar-Choon1,4
1Postgraduate Program in Clinical Biochemistry,
Hasanuddin University, Indonesia
2Prodia Stemcell Indonesia
3Prodia Clinical Laboratory
4National University of Singapore
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Abstract: Ischemic stroke remains a major health problem associated with high mortality
and severe morbidity. The challenge of treatment is to understand the process
leading to endogenous neurorepair mechanism to ischemic stroke. This study
tested the hypothesis that VSEL, SDF-1 and BDNF have important roles in
the process endogenous neurorepair in response to ischemic stroke. Studies
indicated an increase in SDF-1 and VSEL within one week of stroke. BDNF
levels tapered after day 15. Together the studies indicated that BDNF levels
were highest when measured within 7 days of stroke onset and decreased
thereafter. SDF-1 and VSEL were highest at between 7 and 15 days of
stroke onset. The findings indicated that SDF-1 could be key for VSEL to be
mobilized as a natural repair process whereas BDNF might be the correlative
response to prevent cell death.
Keywords: SDF-1, BDNF, VSEL, Ischaemic stroke.
Cancer Vaccines: Bench to Bedside
doi: 10.13052/ijts2246-8765.2016.003
Neha Tuli†,1, Rachana Maniyar†,1, Robert Bednarczyk1,
Ghada Ben Rahoma1, Sarnath Singh1, Jan Geliebter1,
Abraham Mittelma1, Marc Wallack1,2, Debabrata Banerjee3
and Raj K. Tiwari1
1Departmentof Microbiology and Immunology, New York Medical College,
Valhalla, NY 10595, USA
2Department of Surgery, Metropolitan Hospital Centre 1901 1st Avenue,
New York, NY 10029, USA
3Department of Pharmacology, Rutgers, The State University of New Jersey,
Piscataway, NJ 08854, USA
†These authors contributed equally to the preparation of this manuscript
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Abstract: The immune system has immense potential in cancer therapy as it is individualized,
precision driven and robust, however, it is associated with challenges
of its own that include immune evasion, development of tolerance and a sustained
tumor rejection response. Recent FDA approval of several checkpoint
inhibitors, anti-CTLA4, anti-PD-1, has re-invigorated cancer immunology by
demonstrating that tolerance to cancer can be broken to induce a sustained
immune response in patients. Active immunization with multivalent tumor
associated antigens (TAA), however, is still a challenge. We have developed
two specific distinct methods to generate multivalent antigens capable of
tumor regression in prostate cancer and melanoma. In prostate cancer,we have
generated specific multivalent peptide mimetics using phage display synthetic
peptide libraries capable of metastatic tumor regression in an animal model. In
melanoma, we have used a vaccinia virus based antigen retrieval technology
to generate a multivalent antigenic vaccine. The antigenic repertoire is well
defined. A protocol for the melanoma vaccine is FDA approved for clinicaltrials. We envision defining the humoral and cellular immune response to
combine our active vaccine strategy with other treatment modalities including
approved checkpoint inhibitors anti-CTLA4 and anti-PD-1. We believe our
vaccine candidates are a new generation of immune-therapeutics that can
prolong cancer free survival and prevent secondary recurrences. Our studies
have challenged the existing paradigms to re-define cancer immunotherapy
that bridges the gap between humoral and cellular immunity by combining
active immune response with negative checkpoint inhibitors thus activating
pre-existing dormant immune response.
Keywords: cancer vaccines, peptide epitopes, vaccinia virus – antigen
retrieval.
Keratinocyte Induced Differentiation of Mesenchymal Stem Cells into Dermal Myofibroblasts: A Role in EffectiveWound Healing
doi: 10.13052/ijts2246-8765.2016.002
Pravin J. Mishra1, Prasun J. Mishra2 and Debabrata Banerjee3
1Intermountain Precision Genomics, Intermountain Healthcare, Dixie Regional
Medical Center 292 South 1470 East, Suite 201 & 301, St. George, UT 84770, USA
2Department of Biochemical and Cellular Pharmacology, Genentech, 1, DNA Way,
South San Francisco, California 94080, USA
3Department of Pharmacology, Robert Wood Johnson Medical School, Graduate
School of Biomedical Sciences, New Brunswick-Piscataway, Rutgers University,
675 Hoes Lane West, Piscataway, NJ 08854. USA
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Abstract: We have previously demonstrated that human mesenchymal stem cells
(hMSCs) migrate toward human keratinocytes as well as toward conditioned
medium from cultured human keratinocytes (KCM) indicating that the hMSCs
respond to signals from keratinocytes [1]. Using fluorescently labeled cells
we now show that in vitro hMSCs appear to surround keratinocytes, and
this organization is recapitulated in vivo. Incubation of hMSCs with KCM
induced dermal myofibroblast like differentiation characterized by expression
of cytoskeletal markers and increased expression of cytokines including
SDF-1, IL-8, IL-6 and CXCL5. Interaction of keratinocytes with hMSCs
appears to be important in the wound healing process. Therapeutic efficacy of
hMSCs in wound healing was examined in two animal models representing
normal and chronic wound healing. Accelerated wound healing was observed when hMSCs and KCM exposed hMSCs (KCMSCs) were injected near
wound site in nude and NOD/SCID mice. Long term follow up of wound
healing revealed that in the hMSC treated wounds there was little evidence of
residual scarring. These dermal myofibroblast like hMSCs add to the wound
healing process. Together, the keratinocyte and hMSCs morphed dermal
myofibroblast like cells as well as the factors secreted by these cells support
wound healing with minimal scarring. The ability of hMSCs to support wound
healing process represents another striking example of the importance of
keratinocyte and hMSCs interplay in the wound microenvironment resulting
in effective wound healing with minimal scarring.
Keywords: Mesenchymal stem cells, keratinocyte conditioned medium,
dermal myofibroblast, cytokine secretion, wound healing, scarring, animal
models.
Immunoassays for Affinity, Avidity and Competition: Methods to Evaluate Monoclonal (and Polyclonal) Antibodies Useful Assays in Development of New Antibodies, Better Understanding of Commercial Antibodies: Their Usefulness in Specific Aims of Research [SOP]
doi: 10.13052/ijts2246-8765.2016.001
Anita Lewis Antes
New Jersey Medical School/Rutgers University/Newark NJ, USA
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Abstract: Enzyme-linked immunosorbent assay (ELISA) remains the most common
method to identify clones of cells during the development of monoclonal
antibodies. This technique is convenient for the rapid screening of large
numbers of clones, subsequent to the fusion of splenocytes of immunized
mice or rabbits with immortal myeloma cells. In general, when screening for
the production of the desired antibody, an antibody to the target protein or
an antibody from another host is generally unavailable. Thus, the standard
test measures reactivity of attachment passively to the target antigen, and
reporting of that attachment using a secondary antibody reporter (sandwich
assay). Slightly better is the ELISA, which reacts the monoclonal within the
cell culture (supernatant) with the antigen and then applies the mix to plates
coated with an antibody against the target, but from another host species. In
this way, the ability of the test clones to capture the target may be examined.
This method is required when preparing antibodies for immunoprecipitation.
The sandwich assay may leave the investigator with as many as 100 or
more prospective clones to further characterize. To identify clones for further
investigation of the target antigen, cell supernatants may be too dilute. Thus,
the expansion of this enormous number of clones for subsequent purification
of the antibodies would be a daunting task. Here we offer EIA-based assays
which simplify the process of excluding the majority of clones which will not
serve the researcher’s requirements. All the assays can be done from a single
stock of each clone of as little as 2 ml volume.
Keywords: ELISA (enzyme linked immune absorbant assay), specificity,
sensitivity, affinity, monoclonal antibody, hybridoma, epitope.
