Day 1 :
- Stem Cell Research
Location: Webinar
Chair
Miriama Sikorová
Charles University, Prague, Czech Republic.
Session Introduction
Idiberto Jose Zotarelli Filho
ABRAN- Brazilian Association of Nutrology, Brazil
Title: Chitosan-Collagen-Genipin Biomatrix presents physico-chemical and biological properties for the preservation of intrinsic characteristics of adipose mesenchymal stem cells at molecular and chromosomal levels for translational therapy
Time : 10:00-10:30
Biography:
Idiberto Jose is affiliated from ABRAN- Brazilian Association of Nutrology, Brazil. He is a recipient of many awards and grants for his valuable
contributions and discoveries in major area of subject research. His international experience includes various programs, contributions and
participation in different countries for diverse fields of study. His research interests reflect in his/her wide range of publications in various
national and international journals.
Abstract:
Introduction: Therapy of artificial tissues and organs has increased the life expectancy of thousands of people,
and one in five over 65 years has already benefited from tissues and organs generated in vitro. Thus, it is
imperative to develop new strategies for the development and optimization of biomatrices and stem cells to
meet demand, which is capable of promoting tissue and organ regeneration, cost amortization, and safe use at
the cellular level. In this context, adipose mesenchymal stem cells (AMSC) have aroused great scientific interest.
Objective: The biocompatibility at the cellular and chromosomal/chromatidic level of chitosan-collagengenipin
biomatrix with human AMSC was analyzed.
Methods: Different concentrations of genipin for matrix establishment were tested by the analysis of the
number of cells, the degradation, and the degree of crosslinking by spectrophotometry. AMSC was obtained
by enzymatic processing of human lipoaspirate and cultured in Petakas (control) and the selected biomatrix
after physicochemical characterization, with and without cryopreservation. The AMSC were characterized
for adhesion and proliferation by conventional optical microscopy, phase contrast and scanning electron
microscopy, immunophenotyping, cell viability with Trypan blue test, chromosomal stability, by conventional
cytogenetic techniques, neoplastic potential by Papanicolaou test, and the capacity of cell differentiation in three
types of tissues, with a statistical approach.
Results: The crosslinked biomatrix with 0.75% v/v genipin was the best for the cell studies. The tests of adhesion,
proliferation, and differentiation capacity showed that the AMSC in contact with the biomatrix maintained
their natural characteristics. The cytogenetic and Papanicolaou tests after different culture conditions in the
biomatrix did not reveal statistically different frequencies of the control cells (cultured without the biomatrix).
Conclusion: The results suggest that the chitosan-collagen-genipin biomatrices with 0.75% v/v genipin are
biocompatible at the cellular and chromatidic/chromosomal level, with adipose mesenchymal stem cells.
Joglekar, N
Loughborough University, UK
Title: Early detection of delayed apoptosis following cryopreservation of MSCs using mass spectrometry
Time : 10:30-11:00
Biography:
Nishant Joglekar is a final year doctoral researcher from Loughborough University, UK. His PhD is focused on addressing the effects of
cryopreservation during the manufacturing of stem cell-based cell therapies, developing a rapid method to identify biomarkers that could be
used to detect dying cells, post-thaw, during manufacturing. Nishant is skilled in cell culture (T cells and stem cells), and numerous other cellbased
techniques including flow cytometry, qPCR, and fluorescence microscopy.
Abstract:
Statement of the Problem: There is currently a growing interest in the use of Mesenchymal Stem Cells
(MSCs) in the development of cell therapies for the treatment of many common conditions. However, during
manufacturing, apoptosis can lead to a loss of function following cryopreservation and remains an area of
concern. Delayed apoptosis in which cells that appear viable immediately post-thaw undergo apoptosis in a
delayed manner is a particular challenge, with no rapid method to identify such cell populations at an early
stage. The aim of this work is to develop a rapid method, using metabolomics, to detect biomarkers, post-thaw,
that could indicate cells undergoing apoptosis.
Methodology & Theoretical Orientation: Initially, cells were cryopreserved using different freezing procedures
expected to have varying impacts on the cells post-thaw (PT). These included changing the amount of DMSO
concentration and freezing rate. The proliferation and viability of the cells, PT, across five days was then
investigated, along with the immunomodulatory properties of MSCs PT, including their ability to reduce T cell
proliferation and induce T cell differentiation into T regulatory cells (Tregs). Liquid Chromatography / Mass
Spectrometry (LC/MS), and Gas Chromatography / Mass Spectrometry (LC/MS) are now being used to detect
apoptotic biomarkers, PT, that could give an indication of cells undergoing apoptosis.
Findings: Using detrimental freezing procedures showed a significant decrease in viability and the proliferation
of the cells PT. However, cryopreservation did not affect the immunomodulatory properties of the MSCs. Initial
preliminary work using GC/MS has shown that healthy MSCs reduce the amount of a specific compound in the
headspace of cell cultures over 24hrs PT.
Conclusion & Significance: While results are only preliminary, the decrease in the compound, PT, could be
used as a biomarker for cell health. This could be used as a rapid quality control step during manufacturing.
Kaixuan Zhao
UniversitKarolinska University Hospital, Swedeny
Title: In Vivo T-Type Ca2+ channel inhibition facilitates maturation of Glucose-Dependent Ca2+ signaling in human iPSC-Derived islets
Time : 11:00-11:30
Biography:
Kaixuan Zhao is a PhD candidate in Medical Science at Karolinska Institutet, Sweden. She is studying the role of voltage-gated Ca2+ channels
in beta cell maturity. Her efforts have resulted in interesting publications in Proc Natl Acad Sci USA, Cell Mol Life Sci and Cell Transplantat.
Abstract:
T-type Ca2+ channels operate in embryonic stem cells, but conduct relatively small Ca2+ currents in
mature human β cells. In certain pathological contexts, e.g., when T-type Ca2+ channels undergo
elevated expression, they mediate exaggerated Ca2+ influx to dissipate β cell maturity. This prompted us to
hypothesize that altered T-type Ca2+ channel activity in human iPSC-derived islet (hiPSC-islet) cells affect
maturation. To test our hypothesis, we transplanted hiPSC-isles into the anterior chamber of the eye (ACE) of
immunodeficient mice, intravitreally infused T-type Ca2+ channel blocker NNC55-0396 and performed in vivo
and ex vivo measurements. In vivo stereomicroscopy showed that transplanted hiPSC-islets underwent initial
adhesion to, gradual integration with and eventual engraftment as well as survival on the iris. In vivo confocal
microscopy revealed that intracameral hiPSC-islets were satisfactorily vascularized and displayed intense light
scattering signals, reflecting the abundance of zinc-insulin crystals inside insulin secretory granules, within
two months post-transplantation. Furthermore, intravitreally-infused NNC55-0396 did not influence the
macromorphology, vascularization and light scattering signals. Interestingly, ex vivo [Ca2+]i measurements
disclosed that intravitreally-infused NNC55-0396 significantly decreased basal [Ca2+]i levels and increased
glucose-stimulated [Ca2+]i responses in intact hiPSC-isles. In conclusion, the present study verifies that the
immunodeficient mouse ACE can serve as a unique site for pharmacological manipulation of in vivo maturation
of hiPSC-islets. These cells can not only be micro-imaged intravitally, noninvasively and longitudinally, but also
retrieved without suffering physical and chemical disturbance for more precise ex vivo studies, as exemplified
here by [Ca2+]i measurements. Importantly, our data demonstrate that inhibition of T-Type Ca2+ channels
facilitates glucose-dependent Ca2+ signaling in hiPSC-islets. These findings are important and support the
notion that altered T-type Ca2+ channel activity may serve as a key signal in hiPSC-islet cell maturation.
Sikorová, M
Charles University, Czech Republic
Title: Cells´ viability changes owing to hypoxia in single culture or co-cultivated leukemic and stem cells
Biography:
Miriama Sikorová is currently a student of the 2nd year of doctoral studies at the 1st Medical Faculty of Charles University in Prague, in the field
of Cell Biology and Pathobiology. She has started her research career while studying at university, where she became enthused for molecular,
but also immunological and cytological research. She is currently engaged in studies of microenvironment of leukemia cells. Cell cultures and
their biochemical, molecular, cytological, microscopic, and no less important statistical analyses take significant proportion of her time. She
always tries to research as deeply as possible, in order to understand broader context. Thanks to her own intense interest, as well as an active
approach, she constantly advances in acquired theoretical and practical knowledge within the given issue.
Abstract:
Preceding researches of hematological malignancies have focused their research on Patient-derived xenograft
models when directly insert patient tumor cells into immunodeficient mice. Culture conditions and other
factors presented in the leukemic microenvironment don´t reflect the in vivo situation and may control behavior
of the cultivated leukemic cells resulting in decrease of their viability and influencing other attributes too. We
aim our project to conquer these drawbacks by testing new approaches for leukemic cell in vitro cultivation
comprising inspection of metabolic, proliferative and morphological changes of several leukemic cell types.
As feeder cells we used human dermal fibroblasts (NHDF) and human mesenchymal stem cells. As leukemic
cells HBL-2 cell line (Human Mantle Cell Lymphoma); SD-1 cell line (acute lymphoblastic leukemia) and UPF
4D cell line (Diffuse large B‑cell lymphoma) were used. Our experiments focused on a single cell cultivation
as well as a co-cultivation of selected leukemic and feeder cells performed simultaneously under hypoxic (1%
O2) and normoxic (20% O2) conditions. Comprehensive examination of all types of leukemic cells revealed
differences in number of cells and a type of energy metabolism, leaning on the form of cultivation, type of used
feeder cells as well as on oxygen concentration.
Generally, the leukemic cell lines more proliferated in co-cultivation with both feeders, however, the oxygen
concentration was decisive. In co-cultivation with NHDF feeder cells, HBL-2 cells´ proliferation was significantly
increased only in hypoxia, while SD-1 cells´ only in normoxia. In case of UPF 4 D cells, hypoxia increased
their proliferation in co-cultivation with both types of feeders. The type of energy metabolism of leukemic
cells changed in favor of glycolysis with decreasing oxygen concentration. In summary, our findings might be
an acquisition for further improvement of the character of leukemic cells in vitro for their subsequent usage in
successful in vivo xenotransplantation.