On 2014 May 12, G L Francis commented:

This paper sought to find a chemically-defined medium to maintain human embryonic stem cells (hESCs) in long-term culture, necessary for expansion of cell numbers to enable production platforms required to provide differentiated cells for the clinical trial of cell-mediated therapeutics and beyond. Although this paper in now six years old the authors raise some important points still relevant today, however, I believe they misinterpreted a key finding for the relative importance of the components of ‘lipid rich albumin’ by underestimating the contribution of the albumin carrier itself. The studies initially utilized KnockOut™ Serum Replacement (KOSR) and then AlbuMAX®, a chromatographically purified lipid-rich bovine serum albumin (BSA) preparation, which they established was the active component in KOSR, moreover, both products are problematic themselves when developing production methods for human clinical products, due to their animal origins . None the less, the importance of the albumin borne lipid components in sustaining pluripotency markers of hESCs, indicating self-renewal over five passages, was correctly established (Fig. 2 & Fig.3). They extend this finding to claim evidence for the exclusive role of lipids, and attribute no role for the associated albumin carrier protein, in supporting the self-renewal of the hESC lines (Fig 3 A-E). It is the latter conclusion I take odds with, and in fact believe the results also support another conclusion - that while lipid components are essential to the action of the lipid-rich albumin preparations used, albumin promotes the action of its lipid ligands, to maximize the response as seen for both KOSR and AlbuMAX®. Indeed careful examination of the data (Fig 3B and Fig3D) reported allows the conclusion that the presence of intact albumin increases the expression of the pluripotency markers TRA-1-60 and NANAOG by around 25%, and only that of OCT4 is unchanged. Also in this series of experiments the authors could have included another control treatment where they mixed the intact AlbuMAX® with trypsin inhibitor before adding the protease trypsin. Next the authors correctly proved that after the repeated passage (X7) of hESCs they actually retained pluripotency and the capacity to differentiate into ectoderm, endoderm, and mesoderm, by displaying the appropriate markers, GFAP & Sox1, Sox17 & Pdx1 (latter not shown), and cardiac Troponin T, respectively (Fig.4A). Differentiation of hESCs passaged under all conditions resulted in a drastic reduction of pluripotency markers Oct4 and NANAOG and the appropriate increase of differentiation marker for each germ layer of the generated embroid bodies (EBs). However, for the AlbuMAX® + Trypsin media passaged hESCs the resulting embroid bodies displayed for each germ layer the relevant markers, but numerically (by inspection) reduced by 47% GFAP and 25% SOX1 (ectoderm), 10% SOX17 and ??% Pdx1 (endoderm), and 42% cardiac Troponin T (mesoderm) compared to EBs derived from hESCs cultured with intact AlbuMAX®. This difference was not addressed by the authors to my knowledge, and if all experimental variables are controlled and assuming these differences are statistically robust - then possibly a more complex role for albumin in facilitating the subsequent differentiation of these cell lines is indicated. Furthermore, the presence of lipid-rich albumin (i.e. BSA present in 10% Fetal Bovine Serum) during hESC differentiation and EB growth suggests the observed marker differences result from earlier genetic or epigenetic modifications to the hESCs during the serial passaging phase. While the possible involvement of other endogenous or exogenous ligands of albumin were considered to no avail, reasonably the authors could not cover the full spectrum of effects that albumin could promote directly or indirectly in mammalian cells, many of which I have reviewed more recently (2010-PMID: 20373019 [PubMed]).<br> I wish to sincerely thank the authors for the wide range of issues they raised in relation to this topic and I hope my comments are of some value to them or others.

On 2014 May 12, G L Francis commented:

This paper sought to find a chemically-defined medium to maintain human embryonic stem cells (hESCs) in long-term culture, necessary for expansion of cell numbers to enable production platforms required to provide differentiated cells for the clinical trial of cell-mediated therapeutics and beyond. Although this paper in now six years old the authors raise some important points still relevant today, however, I believe they misinterpreted a key finding for the relative importance of the components of ‘lipid rich albumin’ by underestimating the contribution of the albumin carrier itself. The studies initially utilized KnockOut™ Serum Replacement (KOSR) and then AlbuMAX®, a chromatographically purified lipid-rich bovine serum albumin (BSA) preparation, which they established was the active component in KOSR, moreover, both products are problematic themselves when developing production methods for human clinical products, due to their animal origins . None the less, the importance of the albumin borne lipid components in sustaining pluripotency markers of hESCs, indicating self-renewal over five passages, was correctly established (Fig. 2 & Fig.3). They extend this finding to claim evidence for the exclusive role of lipids, and attribute no role for the associated albumin carrier protein, in supporting the self-renewal of the hESC lines (Fig 3 A-E). It is the latter conclusion I take odds with, and in fact believe the results also support another conclusion - that while lipid components are essential to the action of the lipid-rich albumin preparations used, albumin promotes the action of its lipid ligands, to maximize the response as seen for both KOSR and AlbuMAX®. Indeed careful examination of the data (Fig 3B and Fig3D) reported allows the conclusion that the presence of intact albumin increases the expression of the pluripotency markers TRA-1-60 and NANAOG by around 25%, and only that of OCT4 is unchanged. Also in this series of experiments the authors could have included another control treatment where they mixed the intact AlbuMAX® with trypsin inhibitor before adding the protease trypsin. Next the authors correctly proved that after the repeated passage (X7) of hESCs they actually retained pluripotency and the capacity to differentiate into ectoderm, endoderm, and mesoderm, by displaying the appropriate markers, GFAP & Sox1, Sox17 & Pdx1 (latter not shown), and cardiac Troponin T, respectively (Fig.4A). Differentiation of hESCs passaged under all conditions resulted in a drastic reduction of pluripotency markers Oct4 and NANAOG and the appropriate increase of differentiation marker for each germ layer of the generated embroid bodies (EBs). However, for the AlbuMAX® + Trypsin media passaged hESCs the resulting embroid bodies displayed for each germ layer the relevant markers, but numerically (by inspection) reduced by 47% GFAP and 25% SOX1 (ectoderm), 10% SOX17 and ??% Pdx1 (endoderm), and 42% cardiac Troponin T (mesoderm) compared to EBs derived from hESCs cultured with intact AlbuMAX®. This difference was not addressed by the authors to my knowledge, and if all experimental variables are controlled and assuming these differences are statistically robust - then possibly a more complex role for albumin in facilitating the subsequent differentiation of these cell lines is indicated. Furthermore, the presence of lipid-rich albumin (i.e. BSA present in 10% Fetal Bovine Serum) during hESC differentiation and EB growth suggests the observed marker differences result from earlier genetic or epigenetic modifications to the hESCs during the serial passaging phase. While the possible involvement of other endogenous or exogenous ligands of albumin were considered to no avail, reasonably the authors could not cover the full spectrum of effects that albumin could promote directly or indirectly in mammalian cells, many of which I have reviewed more recently (2010-PMID: 20373019 [PubMed]).<br> I wish to sincerely thank the authors for the wide range of issues they raised in relation to this topic and I hope my comments are of some value to them or others.