TY - JOUR
T1 - Encapsulation of murine hematopoietic stem and progenitor cells in a thiol-crosslinked maleimide-functionalized gelatin hydrogel
AU - Gilchrist, Aidan E.
AU - Serrano, Julio F.
AU - Ngo, Mai T.
AU - Hrnjak, Zona
AU - Kim, Sanha
AU - Harley, Brendan A.C.
N1 - The authors would like to acknowledge Dr. Barbara Pilas and Barbara Balhan of the Roy J. Carver Biotechnology Center (Flow Cytometry Facility, UIUC) for assistance with bone marrow cell isolation and flow cytometry. Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Numbers R01 DK099528 (B.A.C.H) and F31 DK117514 (A.E.G.), the National Cancer Institute of the National Institutes of Health under Award Number R01 CA197488 and R01 CA256481 (B.A.C.H.), as well as the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Numbers R21 EB018481 (B.A.C.H.) and T32 EB019944 (A.E.G.). The authors also acknowledge funding from the National Science Foundation Graduate Research Fellowship (DGE 1144245, M.T.N.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the NSF. The authors are also grateful for additional funding provided by the Department of Chemical & Biomolecular Engineering and the Institute for Genomic Biology at the University of Illinois at Urbana-Champaign.
The authors would like to acknowledge Dr. Barbara Pilas and Barbara Balhan of the Roy J. Carver Biotechnology Center (Flow Cytometry Facility, UIUC) for assistance with bone marrow cell isolation and flow cytometry. Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Numbers R01 DK099528 (B.A.C.H) and F31 DK117514 (A.E.G.), the National Cancer Institute of the National Institutes of Health under Award Number R01 CA197488 and R01 CA256481 (B.A.C.H.), as well as the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Numbers R21 EB018481 (B.A.C.H.) and T32 EB019944 (A.E.G.). The authors also acknowledge funding from the National Science Foundation Graduate Research Fellowship (DGE 1144245, M.T.N.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the NSF. The authors are also grateful for additional funding provided by the Department of Chemical & Biomolecular Engineering and the Institute for Genomic Biology at the University of Illinois at Urbana-Champaign .
PY - 2021/9/1
Y1 - 2021/9/1
N2 - Biomaterial platforms are an integral part of stem cell biomanufacturing protocols. The collective biophysical, biochemical, and cellular cues of the stem cell niche microenvironment play an important role in regulating stem cell fate decisions. Three-dimensional (3D) culture of stem cells within biomaterials provides a route to present biophysical and biochemical stimuli through cell-matrix interactions and cell-cell interactions via secreted biomolecules. Herein, we describe a maleimide-functionalized gelatin (GelMAL) hydrogel that can be crosslinked via thiol-Michael addition click reaction for the encapsulation of sensitive stem cell populations. The maleimide functional units along the gelatin backbone enables gelation via the addition of a dithiol crosslinker without requiring external stimuli (e.g., UV light, photoinitiator), thereby reducing reactive oxide species generation. Additionally, the versatility of crosslinker selection enables easy insertion of thiol-containing bioactive or bioinert motifs. Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) were encapsulated in GelMAL, with mechanical properties tuned to mimic the in vivo bone marrow niche. We report the insertion of a cleavable peptide crosslinker that can be degraded by the proteolytic action of Sortase A, a mammalian-inert enzyme. Notably, Sortase A exposure preserves stem cell surface markers, which are an essential metric of hematopoietic activity used in immunophenotyping. This novel GelMAL system enables a route to produce artificial stem cell niches with tunable biophysical properties, intrinsic cell-interaction motifs, and orthogonal addition of bioactive crosslinks. Statement of significance: We describe a maleimide-functionalized gelatin hydrogel that can be crosslinked via a thiol-maleimide mediated click reaction to form a stable hydrogel without the production of reactive oxygen species typical in light-based crosslinking. The mechanical properties can be tuned to match the in vivo bone marrow microenvironment for hematopoietic stem cell culture. Additionally, we report inclusion of a peptide crosslinker that can be cleaved via the proteolytic action of Sortase A and show that Sortase A exposure does not degrade sensitive surface marker expression patterns. Together, this approach reduces stem cell exposure to reactive oxygen species during hydrogel gelation and enables post-culture quantitative assessment of stem cell phenotype.
AB - Biomaterial platforms are an integral part of stem cell biomanufacturing protocols. The collective biophysical, biochemical, and cellular cues of the stem cell niche microenvironment play an important role in regulating stem cell fate decisions. Three-dimensional (3D) culture of stem cells within biomaterials provides a route to present biophysical and biochemical stimuli through cell-matrix interactions and cell-cell interactions via secreted biomolecules. Herein, we describe a maleimide-functionalized gelatin (GelMAL) hydrogel that can be crosslinked via thiol-Michael addition click reaction for the encapsulation of sensitive stem cell populations. The maleimide functional units along the gelatin backbone enables gelation via the addition of a dithiol crosslinker without requiring external stimuli (e.g., UV light, photoinitiator), thereby reducing reactive oxide species generation. Additionally, the versatility of crosslinker selection enables easy insertion of thiol-containing bioactive or bioinert motifs. Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) were encapsulated in GelMAL, with mechanical properties tuned to mimic the in vivo bone marrow niche. We report the insertion of a cleavable peptide crosslinker that can be degraded by the proteolytic action of Sortase A, a mammalian-inert enzyme. Notably, Sortase A exposure preserves stem cell surface markers, which are an essential metric of hematopoietic activity used in immunophenotyping. This novel GelMAL system enables a route to produce artificial stem cell niches with tunable biophysical properties, intrinsic cell-interaction motifs, and orthogonal addition of bioactive crosslinks. Statement of significance: We describe a maleimide-functionalized gelatin hydrogel that can be crosslinked via a thiol-maleimide mediated click reaction to form a stable hydrogel without the production of reactive oxygen species typical in light-based crosslinking. The mechanical properties can be tuned to match the in vivo bone marrow microenvironment for hematopoietic stem cell culture. Additionally, we report inclusion of a peptide crosslinker that can be cleaved via the proteolytic action of Sortase A and show that Sortase A exposure does not degrade sensitive surface marker expression patterns. Together, this approach reduces stem cell exposure to reactive oxygen species during hydrogel gelation and enables post-culture quantitative assessment of stem cell phenotype.
KW - Gelatin hydrogels
KW - Hematopoietic stem cell
KW - Reactive oxygen species
KW - Stem cells
UR - http://www.scopus.com/inward/record.url?scp=85110075399&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85110075399&partnerID=8YFLogxK
U2 - 10.1016/j.actbio.2021.06.028
DO - 10.1016/j.actbio.2021.06.028
M3 - Article
C2 - 34161871
AN - SCOPUS:85110075399
SN - 1742-7061
VL - 131
SP - 138
EP - 148
JO - Acta Biomaterialia
JF - Acta Biomaterialia
ER -