Efficient differentiation of beige adipocytes from adult human adipose-derived stem/stromal cells

Abstract Beige adipocytes (also known as brite adipocytes) have significant utility for numerous applications, such as drug screening, cell therapy, and disease modelling. However, a high-efficiency protocol from human adult adipose-derived stem/stromal stem cells (ADSC) has not been described. The protocol described here achieves beige adipocyte purities of >92% in a fully-defined, serum-free media cocktail, which enables these downstream applications. This method provides a significant leap forward over previously described, serum-based protocols that were inconsistent and inefficient.

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Comparative Analysis of Mesenchymal Stem Cells Cultivated in Serum Free Media

10.21203/rs.3.rs-1179163/v1 ◽

2022 ◽

Author(s):

Joo Youn Lee ◽

Min Hee Kang ◽

Ji Eun Jang ◽

Jeong Eon Lee ◽

Yuyeong Yang ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Therapy ◽

Mass Production ◽

Differential Expression Analysis ◽

Population Doubling ◽

Animal Origin ◽

Population Doubling Time ◽

Serum Free ◽

Free Media

Abstract Stem cells are attractive candidates for the regeneration of tissue and organ. Mesenchymal stem cells (MSCs) have been extensively investigated for their potential applications in regenerative medicine and cell therapy. For developing effective stem cell therapy, the mass production of consistent quality cells is required. The cell culture medium is the most critical aspect of the mass production of qualified stem cells. Classically, fetal bovine serum (FBS) has been used as a culture supplement for MSCs. Due to the undefined and heterologous composition of animal origin components in FBS, efforts to replace animal-derived components with non-animal-derived substances led to safe serum free media (SFM). Adipose derived mesenchymal stem cells (ADSCs) cultivated in SFM provided a more stable population doubling time (PDT) to later passage and more cells in a shorter time compared to FBS containing media. ADSCs cultivated in SFM had lower cellular senescence, lower immunogenicity, and higher genetic stability than ADSCs cultivated in FBS containing media. Differential expression analysis of mRNAs and proteins showed that the expression of genes related with apoptosis, immune response, and inflammatory response were significantly up-regulated in ADSCs cultivated in FBS containing media. ADSCs cultivated in SFM showed similar therapeutic efficacy in an acute pancreatitis mouse model to ADSCs cultivated in FBS containing media. Consideration of clinical trials, not only pre-clinical trial, suggests that cultivation of MSCs using SFM might offer more safe cell therapeutics as well as repeated administration due to low immunogenicity.

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CD34+ Cobblestone Area-Forming Cells Are a Valuable Indicator to Screen the Soluble Inhibitors of Hematopoiesis in the Serum of Aplastic Anemia and MDS Patients.

Blood ◽

10.1182/blood.v114.22.1439.1439 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1439-1439

Author(s):

Shohei Kikuchi ◽

Masayoshi Kobune ◽

Kazuyuki Murase ◽

Satoshi Iyama ◽

Tsutomu Sato ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Aplastic Anemia ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Serum Free ◽

Free Media ◽

Human Stromal Cells ◽

Stromal Layer

Abstract Abstract 1439 Poster Board I-462 We have previously shown that cobblestone area (CA) could be observed underneath human stromal cells 2 weeks after coculture in serum free condition. However, it was difficult to evaluate 5 week CA-forming cells (CAFC) which is a surrogate indicator of hematopoietic stem cells because human stromal cells could not survive more than 4 weeks in a serum free media, X-VIVO10. In the present study, we optimized serum free media, combination of cytokines and chemical agents such as stem cell factor (SCF), thrombopoietin (TPO), flt3 ligand (FL) with or without delta like protein 4 (DLL4) and GSK inhibitor. In addition, we added the serum derived from aplastic anemia and MDS patients in this coculture system and evaluated the effect of serum on in vitro hematopoiesis. We found that certain serum free media could maintain the layer of human stromal cells more than 8 weeks. By using this serum free media, bone marrow (BM) CD34+ cells could be cocultured for 8 weeks without disruption of the human stromal layer. Five week CAFC could be observed in all condition of cytokine combination with or without chemical inhibitor. However, because cytoplasmic appearance of cells in some CAs is quite irregular without DLL4 or GSK inhibitor, we conducted immunohistochemical staining by using FITC-labeled anti human CD34, CD11b or CD33 antibodies. Surprisingly, we found that CD34+CAFCs were exclusively observed in the presence of DLL4 or GSK inhibitor. Otherwise, CAs was composed of CD11b+/CD45+ hematopoietic cells. Moreover, NOD/SCID repopulating cells were detected in the coculture containing CD34+CAFCs, suggesting that CD34+CAFCs were a valuable indicator of hematopoietic stem cells (HSCs). Using CD34+CAFCs as a surrogate maker of HSCs, we assessed whether the serum derived from patients of aplastic anemia and MDS could inhibit formation of CD34+CAFC. Notably, some of patient's serum could reduce the number of CD34+CAFCs. These results indicated the possibility that CD34+CAFCs underneath human stromal layer could be useful for screening certain soluble factors to inhibit hematopoiesis in aplastic anemia and MDS. Disclosures No relevant conflicts of interest to declare.

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Expansion and characterization of bone marrow derived human mesenchymal stromal cells in serum-free conditions

Scientific Reports ◽

10.1038/s41598-021-83088-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Samatha Bhat ◽

Pachaiyappan Viswanathan ◽

Shashank Chandanala ◽

S. Jyothi Prasanna ◽

Raviraja N. Seetharam

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Population Doubling ◽

Seeding Density ◽

Population Doubling Time ◽

Differentiation Potential ◽

Serum Free ◽

Safety Issues ◽

Free Media

AbstractBone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.

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Human Mesenchymal Stromal Cells Unveil an Unexpected Differentiation Potential toward the Dopaminergic Neuronal Lineage

International Journal of Molecular Sciences ◽

10.3390/ijms21186589 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6589

Author(s):

Giulia Gaggi ◽

Andrea Di Credico ◽

Pascal Izzicupo ◽

Francesco Alviano ◽

Michele Di Mauro ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Dopaminergic Neurons ◽

High Efficiency ◽

Ethical Issues ◽

Differentiation Potential ◽

Homogeneous Population ◽

Human Mesenchymal Stromal Cells ◽

Midbrain Dopaminergic Neurons

Degeneration of dopaminergic neurons represents the cause of many neurodegenerative diseases, with increasing incidence worldwide. The replacement of dead cells with new healthy ones may represent an appealing therapeutic approach to these pathologies, but currently, only pluripotent stem cells can generate dopaminergic neurons with high efficiency. However, with the use of these cells arises safety and/or ethical issues. Human mesenchymal stromal cells (hFM-MSCs) are perinatal stem cells that can be easily isolated from the amniochorionic membrane after delivery. Generally considered multipotent, their real differentiative potential is not completely elucidated. The aim of this study was to analyze their stemness characteristics and to evaluate whether they may overcome their mesenchymal fate, generating dopaminergic neurons. We demonstrated that hFM-MSCs expressed embryonal genes OCT4, NANOG, SOX2, KLF4, OVOL1, and ESG1, suggesting they have some features of pluripotency. Moreover, hFM-MSCs that underwent a dopaminergic differentiation protocol gradually increased the transcription of dopaminergic markers LMX1b, NURR1, PITX3, and DAT. We finally obtained a homogeneous population of cells resembling the morphology of primary midbrain dopaminergic neurons that expressed the functional dopaminergic markers TH, DAT, and Nurr1. In conclusion, our results suggested that hFM-MSCs retain the expression of pluripotency genes and are able to differentiate not only into mesodermal cells, but also into neuroectodermal dopaminergic neuron-like cells.

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Human bone marrow-derived mesenchymal stromal cells cultured in serum-free media demonstrate enhanced antifibrotic abilities via prolonged survival and robust regulatory T cell induction in murine bleomycin-induced pulmonary fibrosis

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02574-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shun Takao ◽

Taku Nakashima ◽

Takeshi Masuda ◽

Masashi Namba ◽

Shinjiro Sakamoto ◽

...

Keyword(s):

Bone Marrow ◽

Pulmonary Fibrosis ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Pulmonary Inflammation ◽

Human Bone ◽

Human Bone Marrow ◽

Distributed Data ◽

Serum Free ◽

Free Media

Abstract Background Mesenchymal stromal cells (MSCs) are a potential therapeutic tool for pulmonary fibrosis. However, ex vivo MSC expansion using serum poses risks of harmful immune responses or unknown pathogen infections in the recipients. Therefore, MSCs cultured in serum-free media (SF-MSCs) are ideal for clinical settings; however, their efficacy in pulmonary fibrosis is unknown. Here, we investigated the effects of SF-MSCs on bleomycin-induced pulmonary inflammation and fibrosis compared to those of MSCs cultured in serum-containing media (S-MSCs). Methods SF-MSCs and S-MSCs were characterized in vitro using RNA sequence analysis. The in vivo kinetics and efficacy of SF-MSC therapy were investigated using a murine model of bleomycin-induced pulmonary fibrosis. For normally distributed data, Student’s t test and one-way repeated measures analysis of variance followed by post hoc Tukey’s test were used for comparison between two groups and multiple groups, respectively. For non-normally distributed data, Kruskal–Wallis and Mann–Whitney U tests were used for comparison between groups, using e Bonferroni’s correction for multiple comparisons. All tests were two-sided, and P < 0.05 was considered statistically significant. Results Serum-free media promoted human bone marrow-derived MSC expansion and improved lung engraftment of intravenously administered MSCs in recipient mice. SF-MSCs inhibited the reduction in serum transforming growth factor-β1 and the increase of interleukin-6 in both the serum and the bronchoalveolar lavage fluid during bleomycin-induced pulmonary fibrosis. SF-MSC administration increased the numbers of regulatory T cells (Tregs) in the blood and lungs more strongly than in S-MSC administration. Furthermore, SF-MSCs demonstrated enhanced antifibrotic effects on bleomycin-induced pulmonary fibrosis, which were diminished by antibody-mediated Treg depletion. Conclusions SF-MSCs significantly suppressed BLM-induced pulmonary inflammation and fibrosis through enhanced induction of Tregs into the lungs and corrected the dysregulated cytokine balance. Therefore, SF-MSCs could be a useful tool for preventing pulmonary fibrosis progression without the demerits of serum use.

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Successful scale up expansion of Wharton's jelly mesenchymal stromal cells in different commercial xeno-free and serum-free media

Cytotherapy ◽

10.1016/j.jcyt.2020.03.162 ◽

2020 ◽

Vol 22 (5) ◽

pp. S94

Author(s):

A. López Fernández ◽

I. Carreras Sánchez ◽

J. Vives

Keyword(s):

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Scale Up ◽

Wharton’S Jelly ◽

Serum Free ◽

Wharton's Jelly ◽

Free Media

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Ex VivoExpansion of Human Mesenchymal Stem Cells in Defined Serum-Free Media

Stem Cells International ◽

10.1155/2012/123030 ◽

2012 ◽

Vol 2012 ◽

pp. 1-21 ◽

Cited By ~ 92

Author(s):

Sunghoon Jung ◽

Krishna M. Panchalingam ◽

Lawrence Rosenberg ◽

Leo A. Behie

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Therapeutic Potential ◽

Culture Media ◽

Human Mesenchymal Stem Cells ◽

Efficient Production ◽

Serum Free ◽

Medium Development ◽

Current Cell ◽

Free Media

Human mesenchymal stem cells (hMSCs) are presently being evaluated for their therapeutic potential in clinical studies to treat various diseases, disorders, and injuries. To date, early-phase studies have indicated that the use of both autologous and allogeneic hMSCs appear to be safe; however, efficacy has not been demonstrated in recent late-stage clinical trials. Optimized cell bioprocessing protocols may enhance the efficacy as well as safety of hMSC therapeutics. Classical media used for generating hMSCs are typically supplemented with ill-defined supplements such as fetal bovine serum (FBS) or human-sourced alternatives. Ideally, culture media are desired to have well-defined serum-free formulations that support the efficient production of hMSCs while maintaining their therapeutic and differentiation capacity. Towards this objective, we review here current cell culture media for hMSCs and discuss medium development strategies.

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