Cell number and chondrogenesis in human mesenchymal stem cell aggregates is affected by the sulfation level of heparin used as a cell coating

Abstract

For particular cell-based therapies, it may be required to culture mesenchymal stem cell (MSC) aggregates with growth factors to promote cell proliferation and/or differentiation. Heparin, a negatively charged glycosaminoglycan (GAG) is known to play an important role in sequestration of positively charged growth factors and, when incorporated within cellular aggregates, could be used to promote local availability of growth factors. We have developed a heparin-based cell coating and we believe that the electrostatic interaction between native heparin and the positively charged growth factors will result in (1) higher cell number in response to fibroblast growth factor-2 (FGF-2) and 2) greater chondrogenic differentiation in response to transforming growth factor-β1 (TGF-β1), compared to a desulfated heparin coating. Results revealed that in the presence of FGF-2, by day 14, heparin-coated MSC aggregates increased in DNA content 8.5 ± 1.6 fold compared to day 1, which was greater than noncoated and desulfated heparin-coated aggregates. In contrast, when cultured in the presence of TGF-β1, by day 21, desulfated heparin-coated aggregates upregulated gene expression of collagen II by 86.5 ± 7.5 fold and collagen X by 37.1 ± 4.7 fold, which was higher than that recorded in the noncoated and heparin-coated aggregates. These observations indicate that this coating technology represents a versatile platform to design MSC culture systems with pairings of GAGs and growth factors that can be tailored to overcome specific challenges in scale-up and culture for MSC-based therapeutics.

Morphological landscapes from high content imaging reveal cytokine priming strategies that enhance mesenchymal stromal cell immunosuppression

“…Additionally, our approach could be used to assess the impact of different manufacturing methods on MSC immunomodulation. Cell culture substrates and biomaterials can be tuned to direct MSC function and morphological profiling could be adapted to screen for biomaterial systems that further enhance MSC function (Hansen et al, 2014;Lei et al, 2016;Ogle et al, 2020;Solorio et al, 2015). It is well established that MSCs lose function and become senescent over the course of ex vivo expansion and identification of soluble cues to include in a defined growth medium could be another application of this morphology-based approach (Karnieli et al, 2017;Wang et al, 2011).…”

Combination of Heparin Binding Peptide and Heparin Cell Surface Coatings for Mesenchymal Stem Cell Spheroid Assembly

“…Biotinylated fully desulfated heparin (Hep-) can also be grafted onto cell surfaces of spheroids and has been visualized for up to 14 days. 30…”

“…This is in accordance with previous findings that that specific GAG sulfation locations (N- and 2-O) are required for FGF-2 binding and activation. 30,32 Therefore, it would be expected that interaction with HEPpep, which is derived from a heparin binding site of FGF-2, would be reduced upon heparin desulfation.…”

“…Desulfation of heparin was performed via acidic methanol treatment for 6 days, as previously published, 29 and conjugated with biotin as per. 30 Biotinylated HEPpep (Biotin-NH 2 -(CH 2 ) 4 -GKRTGQYKLG-NH 2 ) and biotinylated Scramble peptide (Biotin-NH 2 -(CH 2 ) 4 -GTYRKKGLQG-NH 2 ) were both purchased from Aapptec, Louisville, KY. Alexa-Fluor 633 conjugation to HEPpep and heparin was performed by EDC coupling in 0.1 M sodium bicarbonate buffer containing 10 mg/mL HEPpep or heparin, 10mM AlexaFluor®−633-hydrazide (Invitrogen), 20 μM EDC. 30 See supplementary information for more details on materials synthesis.…”

Morphological Landscapes from High Content Imaging Identify Optimal Priming Strategies that Enhance MSC Immunosuppression

“…Cell culture substrates and biomaterials can be tuned to direct MSC function and morphological profiling could be adapted to screen for biomaterial systems that further enhance MSC function. [69][70][71][72] It is well established that MSCs lose function and become senescent over the course of ex vivo expansion and identification of soluble cues to include in defined growth medium could be another application of this morphology-based approach. 73,74 Additionally, cryopreserved MSCs undergo a recovery period post-thaw, during which their function is impaired.…”