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18 pages, 2992 KiB

Open AccessEditor’s ChoiceArticle

Enhanced Expansion of Human Pluripotent Stem Cells and Somatic Cell Reprogramming Using Defined and Xeno-Free Culture Conditions

by Suraj Timilsina, Kaitlyn Faith McCandliss, Evan Trivedi and Luis G. Villa-Diaz

Bioengineering 2023, 10(9), 999; https://doi.org/10.3390/bioengineering10090999 - 24 Aug 2023

Viewed by 1094

Abstract

Human embryonic stem cells and induced pluripotent stem cells (hPSC) have an unprecedented opportunity to revolutionize the fields of developmental biology as well as tissue engineering and regenerative medicine. However, their applications have been significantly limited by the lack of chemically defined and [...] Read more.

Human embryonic stem cells and induced pluripotent stem cells (hPSC) have an unprecedented opportunity to revolutionize the fields of developmental biology as well as tissue engineering and regenerative medicine. However, their applications have been significantly limited by the lack of chemically defined and xeno-free culture conditions. The demand for the high-quality and scaled-up production of cells for use in both research and clinical studies underscores the need to develop tools that will simplify the in vitro culture process while reducing the variables. Here, we describe a systematic study to identify the optimal conditions for the initial cell attachment of hPSC to tissue culture dishes grafted with polymers of N-(3-Sulfopropyl)-N-Methacryloxyethyl-N, N-Dimethylammoniun Betaine (PMEDSAH) in combination with chemically defined and xeno-free culture media. After testing multiple supplements and chemicals, we identified that pre-conditioning of PMEDSAH grafted plates with 10% human serum (HS) supported the initial cell attachment, which allowed for the long-term culture and maintenance of hPSC compared to cells cultured on Matrigel-coated plates. Using this culture condition, a 2.1-fold increase in the expansion of hPSC was observed without chromosomal abnormalities. Furthermore, this culture condition supported a higher reprogramming efficiency (0.37% vs. 0.22%; p < 0.0068) of somatic cells into induced pluripotent stem cells compared to the non-defined culture conditions. This defined and xeno-free hPSC culture condition may be used in obtaining the large populations of hPSC and patient-derived iPSC required for many applications in regenerative and translational medicine. Full article

(This article belongs to the Special Issue Stem Cell Bioprocessing and Tissue Reconstruction)

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24 pages, 3931 KiB

Open AccessArticle

Identification of a Novel Wnt Antagonist Based Therapeutic and Diagnostic Target for Alzheimer’s Disease Using a Stem Cell-Derived Model

by Manasi Patil, Naisarg Gamit, Arun Dharmarajan, Gautam Sethi and Sudha Warrier

Bioengineering 2023, 10(2), 192; https://doi.org/10.3390/bioengineering10020192 - 02 Feb 2023

Cited by 4 | Viewed by 2150

Abstract

Currently, all the existing treatments for Alzheimer’s disease (AD) fail to stall progression due to longer duration of time between onset of the symptoms and diagnosis of the disease, raising the necessity of effective diagnostics and novel treatment. Specific molecular regulation of the [...] Read more.

Currently, all the existing treatments for Alzheimer’s disease (AD) fail to stall progression due to longer duration of time between onset of the symptoms and diagnosis of the disease, raising the necessity of effective diagnostics and novel treatment. Specific molecular regulation of the onset and progression of disease is not yet elucidated. This warranted investigation of the role of Wnt signaling regulators which are thought to be involved in neurogenesis. The AD model was established using amyloid beta (Aβ) in human mesenchymal stem cells derived from amniotic membranes which were differentiated into neuronal cell types. In vivo studies were carried out with Aβ or a Wnt antagonist, AD201, belonging to the sFRP family. We further created an AD201-knockdown in vitro model to determine the role of Wnt antagonism. BACE1 upregulation, ChAT and α7nAChR downregulation with synapse and functionality loss with increases in ROS confirmed the neurodegeneration. Reduced β-catenin and increased AD201 expression indicated Wnt/canonical pathway inhibition. Similar results were exhibited in the in vivo study along with AD-associated behavioural and molecular changes. AD201-knockdown rescued neurons from Aβ-induced toxicity. We demonstrated for the first time a role of AD201 in Alzheimer’s disease manifestation, which indicates a promising disease target and biomarker. Full article

(This article belongs to the Special Issue Stem Cell Bioprocessing and Tissue Reconstruction)

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13 pages, 3567 KiB

Open AccessArticle

Effect of Biodentine on Odonto/Osteogenic Differentiation of Human Dental Pulp Stem Cells

by Xuerong Wang, Yixin Cai, Min Zhang, Junchen Xu, Chengfei Zhang and Jin Li

Bioengineering 2023, 10(1), 12; https://doi.org/10.3390/bioengineering10010012 - 21 Dec 2022

Cited by 1 | Viewed by 1906

Abstract

This study aims to compare the biological characteristics of human dental pulp stem cells (hDPSCs) isolated from different-aged populations and examine the effects of Biodentine on proliferation and odonto/osteogenic differentiation of hDPSCs isolated from the elderly in vitro. hDPSCs were isolated from three [...] Read more.

This study aims to compare the biological characteristics of human dental pulp stem cells (hDPSCs) isolated from different-aged populations and examine the effects of Biodentine on proliferation and odonto/osteogenic differentiation of hDPSCs isolated from the elderly in vitro. hDPSCs were isolated from three different-aged populations: group A (≤18 years old), group B (19–59 years old), and group C (≥60 years old). The adhesion, proliferation, odonto/osteogenesis, and senescence were compared. The optimal concentration of aqueous Biodentine extract was determined by CCK-8 assay, alkaline phosphatase (ALP), and alizarin red staining (ARS). The effect of Biodentine on odonto/osteogenic gene and protein expression of hDPSCs in each group was evaluated by quantitative real-time PCR (QRT-PCR) and Western blot. hDPSCs were successfully isolated from three different-aged populations. Flow cytometry revealed that all isolated hDPSCs were positive for CD73 (>90%), CD90 (>90%), CD146 (<30%), and negative for CD45 (<1%). There existed an age-related decline in proliferation, odonto/osteogenic gene expression, and S-phase fraction (p < 0.05), an increase in senescence genes and p21 and p16 expression, and time needed for cell adhesion. Biodentine promoted hDPSC proliferation and mineralization in each group, particularly at a concentration of 0.2 mg/mL. Biodentine markedly enhanced odonto/osteogenesis-related gene and protein expression in each group (p < 0.05). hDPSCs can be obtained from populations of all ages. Though there is an age-related decline in their biological properties, hDPSCs from the elderly still maintain certain proliferation and multidirectional differentiation abilities. Biodentine can significantly promote the proliferation and odonto/osteogenic differentiation of hDPSCs isolated from the elderly over 60 years old, which could be considered a pulp capping material for vital pulp therapy in the elderly. Nevertheless, the efficacy of Biodentine in clinical application has to be further studied. Full article

(This article belongs to the Special Issue Stem Cell Bioprocessing and Tissue Reconstruction)

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14 pages, 7698 KiB

Open AccessArticle

Effect of Rho–Associated Kinase Inhibitor on Growth Behaviors of Human Induced Pluripotent Stem Cells in Suspension Culture

by Takaki Matsumoto, Mee-Hae Kim and Masahiro Kino-oka

Bioengineering 2022, 9(11), 613; https://doi.org/10.3390/bioengineering9110613 - 25 Oct 2022

Cited by 3 | Viewed by 2308

Abstract

Rho–associated protein kinase (ROCK) inhibitors are used for the survival of single-dissociated human induced pluripotent stem cells (hiPSCs); however, their effects on the growth behaviors of hiPSCs in suspension culture are unexplored. Therefore, we investigated the effect of ROCK inhibitor on growth behaviors [...] Read more.

Rho–associated protein kinase (ROCK) inhibitors are used for the survival of single-dissociated human induced pluripotent stem cells (hiPSCs); however, their effects on the growth behaviors of hiPSCs in suspension culture are unexplored. Therefore, we investigated the effect of ROCK inhibitor on growth behaviors of two hiPSC lines (Tic and 1383D2) with different formation of aggregate that attached between single cells in suspension culture. The apparent specific growth rate by long-term exposure to Y-27632, a ROCK inhibitor, was maintained throughout the culture. Long-term exposure to ROCK inhibitor led to an increase in cell division throughout the culture in both lines. Immunofluorescence staining confirmed that hiPSCs forming spherical aggregates showed localization of collagen type I on its periphery. In addition, phosphorylated myosin (pMLC) was localized at the periphery in culture under short-term exposure to ROCK inhibitor, whereas pMLC was not detected at whole the aggregate in culture under long-term exposure. Scanning electron microscopy indicated that long-term exposure to ROCK inhibitor blocked the structural alteration on the surface of cell aggregates. These results indicate that pMLC inhibition by long-term ROCK inhibition leads to enhanced growth abilities of hiPSCs in suspension culture by maintaining the structures of extracellular matrices. Full article

(This article belongs to the Special Issue Stem Cell Bioprocessing and Tissue Reconstruction)

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Review

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18 pages, 2665 KiB

Open AccessReview

Unlocking the Potential of Stem Cell Microenvironments In Vitro

by Chiara Scodellaro, Raquel R. Pina, Frederico Castelo Ferreira, Paola Sanjuan-Alberte and Tiago G. Fernandes

Bioengineering 2024, 11(3), 289; https://doi.org/10.3390/bioengineering11030289 - 19 Mar 2024

Viewed by 800

Abstract

The field of regenerative medicine has recently witnessed groundbreaking advancements that hold immense promise for treating a wide range of diseases and injuries. At the forefront of this revolutionary progress are stem cells. Stem cells typically reside in specialized environments in vivo, known [...] Read more.

The field of regenerative medicine has recently witnessed groundbreaking advancements that hold immense promise for treating a wide range of diseases and injuries. At the forefront of this revolutionary progress are stem cells. Stem cells typically reside in specialized environments in vivo, known as microenvironments or niches, which play critical roles in regulating stem cell behavior and determining their fate. Therefore, understanding the complex microenvironments that surround stem cells is crucial for advancing treatment options in regenerative medicine and tissue engineering applications. Several research articles have made significant contributions to this field by exploring the interactions between stem cells and their surrounding niches, investigating the influence of biomechanical and biochemical cues, and developing innovative strategies for tissue regeneration. This review highlights the key findings and contributions of these studies, shedding light on the diverse applications that may arise from the understanding of stem cell microenvironments, thus harnessing the power of these microenvironments to transform the landscape of medicine and offer new avenues for regenerative therapies. Full article

(This article belongs to the Special Issue Stem Cell Bioprocessing and Tissue Reconstruction)

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22 pages, 2062 KiB

Open AccessReview

The Exciting Realities and Possibilities of iPS-Derived Cardiomyocytes

by Fuga Takahashi, Praneel Patel, Takahiro Kitsuka and Kenichi Arai

Bioengineering 2023, 10(2), 237; https://doi.org/10.3390/bioengineering10020237 - 10 Feb 2023

Cited by 4 | Viewed by 2474

Abstract

Induced pluripotent stem cells (iPSCs) have become a prevalent topic after their discovery, advertised as an ethical alternative to embryonic stem cells (ESCs). Due to their ability to differentiate into several kinds of cells, including cardiomyocytes, researchers quickly realized the potential for differentiated [...] Read more.

Induced pluripotent stem cells (iPSCs) have become a prevalent topic after their discovery, advertised as an ethical alternative to embryonic stem cells (ESCs). Due to their ability to differentiate into several kinds of cells, including cardiomyocytes, researchers quickly realized the potential for differentiated cardiomyocytes to be used in the treatment of heart failure, a research area with few alternatives. This paper discusses the differentiation process for human iPSC-derived cardiomyocytes and the possible applications of said cells while answering some questions regarding ethical issues. Full article

(This article belongs to the Special Issue Stem Cell Bioprocessing and Tissue Reconstruction)

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24 pages, 2624 KiB

Open AccessReview

Recent Advances in Cell Sheet Engineering: From Fabrication to Clinical Translation

by Parichut Thummarati, Wanida Laiwattanapaisal, Rikiya Nitta, Megumi Fukuda, Artchaya Hassametto and Masahiro Kino-oka

Bioengineering 2023, 10(2), 211; https://doi.org/10.3390/bioengineering10020211 - 06 Feb 2023

Cited by 6 | Viewed by 3802

Abstract

Cell sheet engineering, a scaffold-free tissue fabrication technique, has proven to be an important breakthrough technology in regenerative medicine. Over the past two decades, the field has developed rapidly in terms of investigating fabrication techniques and multipurpose applications in regenerative medicine and biological [...] Read more.

Cell sheet engineering, a scaffold-free tissue fabrication technique, has proven to be an important breakthrough technology in regenerative medicine. Over the past two decades, the field has developed rapidly in terms of investigating fabrication techniques and multipurpose applications in regenerative medicine and biological research. This review highlights the most important achievements in cell sheet engineering to date. We first discuss cell sheet harvesting systems, which have been introduced in temperature-responsive surfaces and other systems to overcome the limitations of conventional cell harvesting methods. In addition, we describe several techniques of cell sheet transfer for preclinical (in vitro and in vivo) and clinical trials. This review also covers cell sheet cryopreservation, which allows short- and long-term storage of cells. Subsequently, we discuss the cell sheet properties of angiogenic cytokines and vasculogenesis. Finally, we discuss updates to various applications, from biological research to clinical translation. We believe that the present review, which shows and compares fundamental technologies and recent advances in cell engineering, can potentially be helpful for new and experienced researchers to promote the further development of tissue engineering in different applications. Full article

(This article belongs to the Special Issue Stem Cell Bioprocessing and Tissue Reconstruction)

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17 pages, 4265 KiB

Open AccessReview

Cell Behavioral Dynamics as a Cue in Optimizing Culture Stabilization in the Bioprocessing of Pluripotent Stem Cells

by Naruchit Thanuthanakhun, Mee-Hae Kim and Masahiro Kino-oka

Bioengineering 2022, 9(11), 669; https://doi.org/10.3390/bioengineering9110669 - 09 Nov 2022

Cited by 2 | Viewed by 1957

Abstract

Pluripotent stem cells (PSCs) are important for future regenerative medicine therapies. However, in the production of PSCs and derivatives, the control of culture-induced fluctuations in the outcome of cell quality remains challenging. A detailed mechanistic understanding of how PSC behaviors are altered in [...] Read more.

Pluripotent stem cells (PSCs) are important for future regenerative medicine therapies. However, in the production of PSCs and derivatives, the control of culture-induced fluctuations in the outcome of cell quality remains challenging. A detailed mechanistic understanding of how PSC behaviors are altered in response to biomechanical microenvironments within a culture is necessary for rational bioprocessing optimization. In this review, we discuss recent insights into the role of cell behavioral and mechanical homeostasis in modulating the states and functions of PSCs during culture processes. We delineate promising ways to manipulate the culture variability through regulating cell behaviors using currently developed tools. Furthermore, we anticipate their potential implementation for designing a culture strategy based on the concept of Waddington’s epigenetic landscape that may provide a feasible solution for tuning the culture quality and stability in the bioprocessing space. Full article

(This article belongs to the Special Issue Stem Cell Bioprocessing and Tissue Reconstruction)

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