Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability, motility, and tropism to brain cancer

Abstract Adult human adipose-derived mesenchymal stem cells (hAMSCs) are multipotent cells, which are abundant, easily collected, and bypass the ethical concerns that plague embryonic stem cells. Their utility and accessibility have led to the rapid development of clinical investigations to explore their autologous and allogeneic cellular-based regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer properties, among others. hAMSCs are typically cultured under ambient conditions with 21% oxygen. However, physiologically, hAMSCs exist in an environment of much lower oxygen tension. Furthermore, hAMSCs cultured in standard conditions have shown limited proliferative and migratory capabilities, as well as limited viability. This study investigated the effects hypoxic culture conditions have on primary intraoperatively derived hAMSCs. hAMSCs cultured under hypoxia (hAMSCs-H) remained multipotent, capable of differentiation into osteogenic, chondrogenic, and adipogenic lineages. In addition, hAMSCs-H grew faster and exhibited less cell death. Furthermore, hAMSCs-H had greater motility than normoxia-cultured hAMSCs and exhibited greater homing ability to glioblastoma (GBM) derived from brain tumor-initiating cells from our patients in vitro and in vivo. Importantly, hAMSCs-H did not transform into tumor-associated fibroblasts in vitro and were not tumorigenic in vivo. Rather, hAMSCs-H promoted the differentiation of brain cancer cells in vitro and in vivo. These findings suggest an alternative culturing technique that can enhance the function of hAMSCs, which may be necessary for their use in the treatment of various pathologies including stroke, myocardial infarction, amyotrophic lateral sclerosis, and GBM.

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Immunomodulative effects of mesenchymal stem cells derived from human embryonic stem cells in vivo and in vitro

Journal of Zhejiang University SCIENCE B ◽

10.1631/jzus.b1000074 ◽

2011 ◽

Vol 12 (1) ◽

pp. 18-27 ◽

Cited By ~ 19

Author(s):

Zhou Tan ◽

Zhong-yuan Su ◽

Rong-rong Wu ◽

Bin Gu ◽

Yu-kan Liu ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem

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Dynamics of anteroposterior axis establishment in a mammalian embryo-like system

10.1101/2021.02.24.432766 ◽

2021 ◽

Author(s):

Kerim Anlaş ◽

Nicola Gritti ◽

David Oriola ◽

Krisztina Arató ◽

Fumio Nakaki ◽

...

Keyword(s):

Stem Cells ◽

Symmetry Breaking ◽

Marker Gene ◽

Embryonic Stem ◽

Culture Conditions ◽

Body Plan ◽

Germ Layer ◽

Mammalian Embryo

1.AbstractIn the mammalian embryo, specification of the anteroposterior (AP) axis demarcates one of the first steps of body plan formation. While this process requires interactions with extra-embryonic tissues in the native embryo, minimal in vitro systems from embryonic stem cells (ESCs) undergo initial AP polarization in the absence of any localized, external cues. This self-organizing potential of stem cells remains not well understood. Here, we study such an initial symmetry breaking event in gastruloids, an established in vitro model for mammalian body plan formation, using the mesodermal marker gene Brachyury or T (Bra/T) to denote the onset of AP axis specification and concomitant germ layer formation. Through aggregate fusion experiments and manipulation of initial culture conditions as well as key developmental signalling pathways, we probe the dynamics of Bra/T polarization. We further conduct single-cell (sc) RNA sequencing of gastruloids at early stages to identify incipient molecular signatures of germ layer commitment and differences between Bra/T+ and Bra/T− populations during as well as after symmetry breaking. Moreover, we transcriptionally compare early development of gastruloids to the mouse embryo and conclude that gastruloids reproducibly undergo AP axis and germ layer specification in a parallel, but distinct manner: While their primed pluripotent cell populations adopt a more mesenchymal state in lieu of an epithelial epiblast-like transcriptome, the emerging mesendodermal lineages in vitro are nevertheless similar to their in vivo equivalents. Altogether, this study provides a comprehensive analysis of self-organized body plan establishment in a minimal in vitro system of early mammalian patterning and highlights the regulative capacity of mESCs, thereby shedding light on underlying principles of axial polarity formation.

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Self-Assembling Scaffolds Supported Long-Term Growth of Human Primed Embryonic Stem Cells and Upregulated Core and Naïve Pluripotent Markers

Cells ◽

10.3390/cells8121650 ◽

2019 ◽

Vol 8 (12) ◽

pp. 1650 ◽

Cited By ~ 4

Author(s):

Christina McKee ◽

Christina Brown ◽

G. Rasul Chaudhry

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Three Dimensional ◽

Embryonic Stem ◽

Extended Period ◽

Culture Conditions ◽

Differentiation Potential ◽

Self Assembling

The maintenance and expansion of human embryonic stem cells (ESCs) in two-dimensional (2-D) culture is technically challenging, requiring routine manipulation and passaging. We developed three-dimensional (3-D) scaffolds to mimic the in vivo microenvironment for stem cell proliferation. The scaffolds were made of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups, which self-assembled via a Michael addition reaction. When primed ESCs (H9 cells) were mixed with PEG polymers, they were encapsulated and grew for an extended period, while maintaining their viability, self-renewal, and differentiation potential both in vitro and in vivo. Three-dimensional (3-D) self-assembling scaffold-grown cells displayed an upregulation of core pluripotency genes, OCT4, NANOG, and SOX2. In addition, the expression of primed markers decreased, while the expression of naïve markers substantially increased. Interestingly, the expression of mechanosensitive genes, YAP and TAZ, was also upregulated. YAP inhibition by Verteporfin abrogated the increased expression of YAP/TAZ as well as core and naïve pluripotent markers. Evidently, the 3-D culture conditions induced the upregulation of makers associated with a naïve state of pluripotency in the primed cells. Overall, our 3-D culture system supported the expansion of a homogenous population of ESCs and should be helpful in advancing their use for cell therapy and regenerative medicine.

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Imaging and Analysis of 3D Tumor Spheroids Enriched for a Cancer Stem Cell Phenotype

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057110376541 ◽

2010 ◽

Vol 15 (7) ◽

pp. 820-829 ◽

Cited By ~ 39

Author(s):

Fredika M. Robertson ◽

Marcia A. Ogasawara ◽

Zaiming Ye ◽

Khoi Chu ◽

Ross Pickei ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem ◽

Chemotherapeutic Agents ◽

Plastic Substrate ◽

Cell Phenotype ◽

Tumor Initiating Cells ◽

Tumor Spheroids ◽

Primary Tumors

Tumors that display a highly metastatic phenotype contain subpopulations of cells that display characteristics similar to embryonic stem cells. These cells exhibit the ability to undergo self-renewal; slowly replicate to retain a nucleoside analog label, leading to their definition as “label-retaining cells”; express specific surface markers such as CD44+/CD24–/low and CD133; and can give rise to cells of different lineages (i.e., they exhibit multipotency). Based on these characteristics, as well as their demonstrated ability to give rise to tumors in vivo, these cells have been defined as tumor-initiating cells (TICs), tumor-propagating cells, or cancer stem cells (CSCs). These cells are highly resistant to chemotherapeutic agents and radiation and are believed to be responsible for the development of both primary tumors and metastatic lesions at sites distant from the primary tumor. Established cancer cell lines contain CSCs, which can be propagated in vitro using defined conditions, to form 3D tumor spheroids. Because the vast majority of studies to identify cancer-associated genes and therapeutic targets use adherent cells grown in 2 dimensions on a plastic substrate, the multicellular composition of these 3D tumor spheroids presents both challenges and opportunities for their imaging and characterization. The authors describe approaches to image and analyze the properties of CSCs within 3D tumor spheroids, which can serve as the basis for defining the gene and protein signatures of CSCs and to develop therapeutic strategies that will effectively target this critically important population of cells that may be responsible for tumor progression.

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383 IDENTIFICATION OF PLURIPOTENCY MARKERS IN SWINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab383 ◽

2010 ◽

Vol 22 (1) ◽

pp. 348

Author(s):

F. R. O. de Barros ◽

M. D. Goissis ◽

M. G. Marques ◽

M. I. Giassetti ◽

F. F. Paula-Lopes ◽

...

Keyword(s):

Stem Cells ◽

In Vitro Culture ◽

Genetic Manipulation ◽

Embryonic Stem ◽

Culture Conditions ◽

Rabbit Serum ◽

Embryonic Fibroblasts ◽

Pluripotency Markers

Embryonic stem cells (ESC) are a useful tool for studying embryonic development, cell differentiation, and genetic manipulation. Moreover, these cells can be applied in cell-based therapies and in vitro organogenesis. The research conducted with human ESC has generated many ethical, moral, and religious considerations by scientists and laymen alike. Therefore, an animal model such as the pig (Sus scrofa) is valuable in overcoming such hurdles because this species holds physiologic parameters similar to humans. In spite of the great biomedical potential of ESC, many difficulties have been faced in maintaining these cells in a pluripotent state in vitro. For this reason, studies to elucidate the mechanisms of in vitro maintenance of undifferentiated ESC are needed to improve the culture of these cells. The objectives of this study were (1) to isolate ESC from in vitro- and in vivo-produced swine blastocysts; (2) to compare 2 in vitro culture conditions to maintain isolated inner cell masses (ICM), murine embryonic fibroblasts (MEF), or Matrigel; and (3) to identify and to compare the expression of the pluripotency markers Nanog, Sox2, and FoxD3 at ESC and in vitro- and in vivo-produced swine blastocysts. In this manner, swine blastocysts were obtained by in vitro maturation and fertilization of oocytes from ovaries collected in abattoirs. Embryos were in vitro cultured for 7 days until blastocyst stage. In addition, in vivo-produced blastocysts were obtained by superovulation followed by AI of gilts (150 days of age). Embryos were collected by post-mortem uterus flushing 5 days after ovulation. In vitro- and in vivo-produced blastocysts were submitted to immunosurgery to isolate the ICM. Briefly, zona pellucida was digested with pronase solution, and embryos were incubated with anti-swine rabbit serum to remove trophoectoderm cells and with guinea-pig complement serum. Resultant ICM (14 and 66 ICM from in vitro- and in vivo-produced blastocysts, respectively) were cultured in stem cells media (GMEM added by 15% FCS, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, and 4 ng mL-1 of basic fibroblast growth factor) over monolayer of irradiated mouse embryonic fibroblasts (MEF) or Matrigel for 3 weeks. No difference was observed between the in vitro culture conditions (MEF and Matrigel) on isolated ICM adhesion. In addition, no difference was verified between in vitro- and in vivo-produced blastocysts on adhesion of cultured ICM. However, no swine ESC was obtained. Gene expression analysis was performed only with whole in vitro- and in vivo-produced blastocysts. Results showed that Nanog and Sox2 were less expressed in in vitro-produced blastocysts. However, the expression of FoxD3, demonstrated in this study for the first time, was similar between groups. Because no ESC lineage was obtained in swine until now, we believe this species has different requirements compared with murine and human. Therefore, more studies are necessary to establish protocols to isolate porcine ESC. Acknowledgments are given to FAPESP (processes 06/58507-0 and 07/51732-0).

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Production of Mesenchymal Stem Cells Through Stem Cell Reprogramming

International Journal of Molecular Sciences ◽

10.3390/ijms20081922 ◽

2019 ◽

Vol 20 (8) ◽

pp. 1922 ◽

Cited By ~ 15

Author(s):

Ahmed Abdal Dayem ◽

Soo Bin Lee ◽

Kyeongseok Kim ◽

Kyung Min Lim ◽

Tak-il Jeon ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Population Heterogeneity ◽

Immunomodulatory Activity ◽

Therapeutic Applications ◽

Proliferation Capacity

Mesenchymal stem cells (MSCs) possess a broad spectrum of therapeutic applications and have been used in clinical trials. MSCs are mainly retrieved from adult or fetal tissues. However, there are many obstacles with the use of tissue-derived MSCs, such as shortages of tissue sources, difficult and invasive retrieval methods, cell population heterogeneity, low purity, cell senescence, and loss of pluripotency and proliferative capacities over continuous passages. Therefore, other methods to obtain high-quality MSCs need to be developed to overcome the limitations of tissue-derived MSCs. Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are considered potent sources for the derivation of MSCs. PSC-derived MSCs (PSC-MSCs) may surpass tissue-derived MSCs in proliferation capacity, immunomodulatory activity, and in vivo therapeutic applications. In this review, we will discuss basic as well as recent protocols for the production of PSC-MSCs and their in vitro and in vivo therapeutic efficacies. A better understanding of the current advances in the production of PSC-MSCs will inspire scientists to devise more efficient differentiation methods that will be a breakthrough in the clinical application of PSC-MSCs.

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Mesenchymal Stem Cells in the Treatment of COVID-19, a Promising Future

Cells ◽

10.3390/cells10102588 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2588

Author(s):

Daniela Gois Beghini ◽

Samuel Iwao Horita ◽

Andrea Henriques-Pons

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Intravenous Injection ◽

Adult Stem Cells ◽

Ethical Issues ◽

Genetic Manipulation ◽

Culture Media ◽

Embryonic Stem

Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in virtually all tissues; they have a potent self-renewal capacity and can differentiate into multiple cell types. They also affect the ambient tissue by the paracrine secretion of numerous factors in vivo, including the induction of other stem cells’ differentiation. In vitro, the culture media supernatant is named secretome and contains soluble molecules and extracellular vesicles that retain potent biological function in tissue regeneration. MSCs are considered safe for human treatment; their use does not involve ethical issues, as embryonic stem cells do not require genetic manipulation as induced pluripotent stem cells, and after intravenous injection, they are mainly found in the lugs. Therefore, these cells are currently being tested in various preclinical and clinical trials for several diseases, including COVID-19. Several affected COVID-19 patients develop induced acute respiratory distress syndrome (ARDS) associated with an uncontrolled inflammatory response. This condition causes extensive damage to the lungs and may leave serious post-COVID-19 sequelae. As the disease may cause systemic alterations, such as thromboembolism and compromised renal and cardiac function, the intravenous injection of MSCs may be a therapeutic alternative against multiple pathological manifestations. In this work, we reviewed the literature about MSCs biology, focusing on their function in pulmonary regeneration and their use in COVID-19 treatment.

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Autologous transplantation of mesenchymal stem cells into the portal vein of the miniature pig; a preliminary experiment for NOTES approach

Perspectives in Surgery ◽

10.33699/pis.2019.98.9.350-355 ◽

2019 ◽

Vol 98 (9) ◽

pp. 350-355

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Therapy ◽

Portal Vein ◽

Liver Parenchyma ◽

Miniature Pig ◽

Hepatic Diseases ◽

Study Results

Introduction: There is evidence that mesenchymal stem cells (MSCs) could trans-differentiate into the liver cells in vitro and in vivo and thus may be used as an unfailing source for stem cell therapy of liver disease. Combination of MSCs (with or without their differentiation in vitro) and minimally invasive procedures as laparoscopy or Natural Orifice Transluminal Endoscopic Surgery (NOTES) represents a chance for many patients waiting for liver transplantation in vain. Methods: Over 30 millions of autologous MSCs at passage 3 were transplanted via the portal vein in an eight months old miniature pig. The deposition of transplanted cells in liver parenchyma was evaluated histologically and the trans-differential potential of CM-DiI labeled cells was assessed by expression of pig albumin using immunofluorescence. Results: Three weeks after transplantation we detected the labeled cells (solitary, small clusters) in all 10 samples (2 samples from each lobe) but no diffuse distribution in the samples. The localization of CM-DiI+ cells was predominantly observed around the portal triads. We also detected the localization of albumin signal in CM-DiI labeled cells. Conclusion: The study results showed that the autologous MSCs (without additional hepatic differentiation in vitro) transplantation through the portal vein led to successful infiltration of intact miniature pig liver parenchyma with detectable in vivo trans-differentiation. NOTES as well as other newly developed surgical approaches in combination with cell therapy seem to be very promising for the treatment of hepatic diseases in near future.

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Differentiation of mesenchymal stem cells from humans and animals into insulin-producing cells: An overview in vitro induction forms

Current Stem Cell Research & Therapy ◽

10.2174/1574888x16666201229124429 ◽

2020 ◽

Vol 16 ◽

Author(s):

Bruna O. S. Câmara ◽

Bruno M. Bertassoli ◽

Natália M. Ocarino ◽

Rogéria Serakides

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Culture Medium ◽

Adipogenic Differentiation ◽

Medium Composition ◽

Cell Therapies ◽

Insulin Producing Cells ◽

Msc Differentiation

The use of stem cells in cell therapies has shown promising results in the treatment of several diseases, including diabetes mellitus, in both humans and animals. Mesenchymal stem cells (MSCs) can be isolated from various locations, including bone marrow, adipose tissues, synovia, muscles, dental pulp, umbilical cords, and the placenta. In vitro, by manipulating the composition of the culture medium or transfection, MSCs can differentiate into several cell lineages, including insulin-producing cells (IPCs). Unlike osteogenic, chondrogenic, and adipogenic differentiation, for which the culture medium and time are similar between studies, studies involving the induction of MSC differentiation in IPCs differ greatly. This divergence is usually evident in relation to the differentiation technique used, the composition of the culture medium, the cultivation time, which can vary from a few hours to several months, and the number of steps to complete differentiation. However, although there is no “gold standard” differentiation medium composition, most prominent studies mention the use of nicotinamide, exedin-4, ß-mercaptoethanol, fibroblast growth factor b (FGFb), and glucose in the culture medium to promote the differentiation of MSCs into IPCs. Therefore, the purpose of this review is to investigate the stages of MSC differentiation into IPCs both in vivo and in vitro, as well as address differentiation techniques and molecular actions and mechanisms by which some substances, such as nicotinamide, exedin-4, ßmercaptoethanol, FGFb, and glucose, participate in the differentiation process.

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Lithium-Doped Biological-Derived Hydroxyapatite Coatings Sustain In Vitro Differentiation of Human Primary Mesenchymal Stem Cells to Osteoblasts

Coatings ◽

10.3390/coatings9120781 ◽

2019 ◽

Vol 9 (12) ◽

pp. 781 ◽

Cited By ~ 4

Author(s):

Paula E. Florian ◽

Liviu Duta ◽

Valentina Grumezescu ◽

Gianina Popescu-Pelin ◽

Andrei C. Popescu ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Doping Agent ◽

Hydroxyapatite Coatings ◽

3D Network ◽

Immunofluorescence Labelling ◽

Good Biocompatibility ◽

Adhesion Process

This study is focused on the adhesion and differentiation of the human primary mesenchymal stem cells (hMSC) to osteoblasts lineage on biological-derived hydroxyapatite (BHA) and lithium-doped BHA (BHA:LiP) coatings synthesized by Pulsed Laser Deposition. An optimum adhesion of the cells on the surface of BHA:LiP coatings compared to control (uncoated Ti) was demonstrated using immunofluorescence labelling of actin and vinculin, two proteins involved in the initiation of the cell adhesion process. BHA:LiP coatings were also found to favor the differentiation of the hMSC towards an osteoblastic phenotype in the presence of osteoinductive medium, as revealed by the evaluation of osteoblast-specific markers, osteocalcin and alkaline phosphatase. Numerous nodules of mineralization secreted from osteoblast cells grown on the surface of BHA:LiP coatings and a 3D network-like organization of cells interconnected into the extracellular matrix were evidenced. These findings highlight the good biocompatibility of the BHA coatings and demonstrate that the use of lithium as a doping agent results in an enhanced osteointegration potential of the synthesized biomaterials, which might therefore represent viable candidates for future in vivo applications.

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