DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

More documents

Recommendations

Info

47 III.4 – Isolation and Proliferation of MSCs from Human Umbilical Cord Isolation of hMSC from the Wharton Jelly on Polyacrylamide Hydrogels According to the objectives of this work (I.4), one of the aims was to establish a protocol for the isolation of mesenchymal stem cells from the umbilical cord matrix (UCM) on a soft substrate, namely using functionalized PA hydrogels. To our knowledge this was never done before, and the main objective was to spare the cells from being cultured on stiff tissue-culture polystyrene (TCPs), which we hypothesize that might narrow the plasticity and stemness of MSCs, due to “substrate memory” phenomena that MSCs have been shown to possess (Tse et al., 2011). Human umbilical cord fragments were plated on different polyacrylamide hydrogels prepared with 10%, 12.5% and 15% Acrylamide (Ac), each bearing distinct degrees of stiffness (Table II). Several formulations were tested, since it was unknown what was the minimum stiffness required for the MSCs to migrate from the umbilical cord fragments to the hydrogels by durotaxis. Furthermore, we tested hydrogels coated only with COL-1 and hydrogels coated with COL-1 plus FN, to find out whether this combination of ECM proteins (COL-1 + FN) might somehow favor the process. What we observed in the first condition (≈7 kPa / COL-1) was that hMSCs would migrate from the umbilical cords fragments (UCF) to the ≈7kPa hydrogels in the first week and form small colonies, but after 2 weeks no proliferation was seen and some cells were as if detaching (Figure 17). In the second condition (≈10 kPa / COL-1 + FN) it is observed in the first week the migration of hMSCs from the UCF and the formation of small colonies but after 2 week there is no further proliferation of those colonies and in the third condition (≈12 kPa / COL-1) happens the same observed in the last 2 conditions. Finally in the last condition (≈12 kPa / COL-1 + FN) in the first week there is also migration of hMSCs from the UCF forming colonies but when compared to the others conditions these colonies are more numerous in cells and after the second week we can clearly observe proliferation to the full confluence point (Figure 17). Hence, 12 kPa / COL-1 + FN hydrogels were used onwards for further isolation of hMSCs from umbilical cord fragments.
≈12 kPa / COL-1 +FN ≈12 kPa / COL-1 ≈10 kPa / COL-1+FN ≈7 kPa / COL-1 48 1 Week 2 Weeks Figure 17 – Colonies of hMSCs isolated in different PA hydrogels from UCM-WJ fragments after 1 week and 2 weeks of fragments plating in the hydrogels with just COL-1 or with COL-1 and FN (n=2). Bar corresponds to 200µm. III.5 - Immunophenotypic characterization of UCM-MSC In order to determine if the cells isolated from the Wharton’s jelly on the PA hydrogels were MSCs, the expression of cell-surface antigens was evaluated at passage 2. As a
Page 1 and 2:
DEPARTAMENTO DE CIÊNCIAS DA VIDA F
Page 3 and 4:
ii Queria agradecer à minha namora
Page 5 and 6:
iv isolation of hUCM-MSCs was ever
Page 7 and 8:
vi Conseguimos optimizar um novo pr
Page 9 and 10:
viii Table of contents Acknowledgem
Page 11 and 12:
x III. 6 - Influence on MSCs specif
Page 13 and 14:
xii Figure 2| Proteins related to t
Page 15 and 16:
xiv the change in distribution with
Page 17 and 18: xvi Cells were stained with anti-B-
Page 19 and 20: xviii List of abbreviations AA - Ac
Page 22: 1 Chapter I Introduction
Page 25 and 26: 4 There are many advantages in usin
Page 27 and 28: 6 only ones MSCs can differentiate
Page 29 and 30: Figure 1. Mechanotransduction in a
Page 31 and 32: 10 1C)(Dogterom et al., 2005). Not
Page 33 and 34: 12 extracellular ligand and the cyt
Page 35 and 36: 14 Figure 3 - The Rigidity Sensing
Page 37 and 38: 16 Figure 5 - Images depicted on A)
Page 39 and 40: 18 Figure 7 - A) Immunofluorescence
Page 41 and 42: 20 (GFAP) (is a class-III intermedi
Page 43 and 44: 22 can specify lineage towards cell
Page 45 and 46: 24 I.3 - Project rationale and expe
Page 47 and 48: 26 Figure 12 - MSCs cultured on 1 a
Page 49 and 50: 28 than that in soft substrate. Thi
Page 52: 31 Chapter II - Materials and Metho
Page 55 and 56: 34 Covance and DakoCytomation, resp
Page 57 and 58: 36 clone SAM1; mouse Pe-Cy7 anti-hu
Page 59 and 60: 38 To functionalize the complete su
Page 62: 41 Chapter III Results
Page 65 and 66: 44 2008) and also as previously est
Page 67: 12.5% Ac PBS Fold increase of hMSCs
Page 71 and 72: 50 Figure 19 - Immunophenotype of U
Page 73 and 74: 52 coated with COL-1 (similar to wh
Page 75 and 76: 54 hydrogels of 1 and 7 kPa (for ce
Page 77: Figure 23 - hMSCs cultured on TCP (
Page 81 and 82: 60 Discussion MSCs are widely used
Page 83 and 84: 62 isolating MSCs on the PA hydroge
Page 85 and 86: 64 2011) and expanded on TCPs for 5
Page 88: 67 Conclusion In conclusion, we opt
Page 91 and 92: 70 Cretu, A., Castagnino, P. & Asso
Page 93 and 94: 72 McBeath, R. et al., 2004. Cell s
Page 95: 74 Wang, N., Butler, J.P., and Ingb
show all

DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?