Increased Intracellular Insulin from Differentiated Stem Cells to Insulin- Producing Cells Incorporating B-Carotene

Research Article

Austin J Anal Pharm Chem 2024; 11(1): 1169.

Increased Intracellular Insulin from Differentiated Stem Cells to Insulin- Producing Cells Incorporating B-Carotene

Francisco Josué Avelar Rodríguez¹; Sanghamitra Khandual²; Jorge Gaona Bernal³; Erika Nahomy Marino Marmolejo¹; Jorge Bravo-Madrigal¹; Flor Yohana Flores Hernández¹*

1Department of Medical and Pharmaceutical Biotechnology of Center for Investigation and Assistance in Technology, México

2Department of Industrial Biotechnology of Center for Investigation and Assistance in Technology, México

3Department of Microbiology and Pathology, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara México

*Corresponding author: Flor Yohana Flores Hernández Center for Investigation and Assistance in Technology, Design for State of Jalisco, C.P. 45019, Zapopan, Jalisco, Mexico. Tel: +52 3333455200, Ext. 1328; Fax: +52 3333455200 1001 Email: fflores@ciatej.mx

Received: March 04, 2024 Accepted: April 10, 2024 Published: April 17, 2024

Abstract

Analyzes the effect of β-Carotene (BC) in the processes of differentiation of adipose tissue Human Mesenchymal Stem Cells (hADMSCs) and dental pulp Human Mesenchymal Stem Cells (hDPMSCs) for obtaining Insulin-Producing Cells (IPCs). The BC present in the methanolic extract was analyzed and quantified by UV-VIS, TLC and HPLC methods.

The cell viability test was done by flow cytometry, a concentration of 1.5 μM of BC or AP extract no cytotoxic effect on stem cells, so this concentration was used for the Human Mesenchymal Stem Cells (hMSCs) differentiation protocols for obtaining IPCs. The differentiation tests were done with the AP extract or β-carotene standard addition which demonstrates the increased cell differentiation percentages by the presence of intracellular insulin. This was more outstanding in the hMSCs that were stimulated with the AP in stage 3 and applied at all stages mainly in hDPMSCs, reaching up to 74% of the insulin-positive population respect to 11% where the molecules were not added. RT-PCR was performed for the level of gene expression like MAFA, PDX1 and NKX6.1 and those are found well expressed on differentiated cells.

This could be relevant in cell therapy in diabetes, where inclusion of AP extracts or BC may improve yield of IPCs.

Keywords: Arthrospira platensis; β-carotene; Differentiation; Insulin-producing cells; Mesenchymal stem cells; Microalgae

Introduction

Diabetes Mellitus (DM) is a chronic metabolic disorder with rapid increase in prevalence, affecting 382 million people worldwide, becoming one of the leading diseases of the 21stcentury [1], DM is the result of inadequate supply of functional β-cells, as these cells are responsible for the production of insulin. Diabetes therapy mainly involves insulin, drugs that enhance insulin secretion or inhibitors of the signaling of endogenous glucose production [2]. However, it is important what kind of cell therapy in diabetes considers the replacement of β-cells derived from human stem cells by the induction of endogenous regeneration through the formation of new cells expressing insulin either by conversion of a differentiated cell type (trans-differentiation) or differentiation of progenitors (neogenesis) in order to cure the disease [3].

Specifically, the use of Human Mesenchymal Stem Cells (hMSCs) has a high potential use in various therapies, due to their multipotent self-renewal capacity as well as potential to differentiate into mesodermal, endodermal and ectodermal lineages. The hMSCs demonstrate expression of specific cell surface molecules such as CD105, CD73 or CD90 and CD44. The use of hMSCs presents several advantages such as the immunomodulatory potential, the reduction of problems related to tumorigenicity and that are found in adult tissues, so that their use may overcome potential ethical issues. Several tissues that contain stem cells and these areas they have been called "niches", such as Human Adipose Tissue (hADMSCs), Human Bone Marrow (hBMMSCs), Human Wharton's Gelatin (hWJMSC), Human Umbilical Cord Blood (hUCBMSC) and Human Dental Pulp (hDPMSCs) [1]. hMSCs can be differentiated into various types of specialized cells such as Insulin-Producing Cells (IPCs). Several protocols for differentiation have been implemented to obtain IPCs, which vary according to the type of mesenchymal stem cell. In general, the protocols are carried out in three stages, where hMSCs are directed towards endoderm, later to endocrine progenitors and finally towards IPCs [4]. Due to the low efficiency of differentiation for obtaining the cells and efficiency of the functionality of these cells, there is a great interest the development of an improved process of in vitro differentiation, but information regarding the factors of the efficient differentiation of hMSCs to β-cells are yet unknown [1]. Specifically, hADMSCs and hDPMSCs are important cell sources and frequently used in the field of tissue engineering and/or regenerative medicine. Due to its easy disponibility, pluripotency capacity and easy proliferation qualities, these cells can be exploited in cell therapy for diabetes to increase the yield of IPCs.

One of the strategies that is followed to increase the efficiency of differentiation protocols of differentiation is the search for new molecules, including those from phycoextracts. Several works focused on the study of the therapeutic activity of BC analyze the inhibition of tumorigenesis in stem cells and discuss the activity of this molecule in the differentiation of these cells by regulating differentiation markers, such as Drosophila Delta-Like 1 Homologue (DLK1) which is a member of the Epidermis Growth Factor (EGF) similar to the family of homeotic proteins and is known to modulate the differentiation signaling in adipocytes and several types of stem cells. However, little is known about the role of this type of extracts and molecules in a process of differentiation of hMSCs towards IPCs cells [5].

Arthrospira Platensis (AP) is a blue-green algae belonging to the family of cyanobacteria, rich in bioactive compounds, such as proteins, lipids, carbohydrates, trace elements (zinc, magnesium, manganese, selenium), riboflavin, tocopherol, and a-linoleic acid, has a 62% amino acid content and is rich in vitamin B12 and it contains a whole spectrum of natural mixed phytopigments. Among them, carotenoids and phycocyanins, some of these compounds have antioxidant activity such as β-Carotene (BC) [6]. In spirulina, BC represents 67–79%, and it has been reported that these protections from photo oxidative damage and BC holds the prime position in provitamin A activity. BC is a known antioxidant and precursor of the retinoic acid molecule of relevance in differentiation processes [5].

AP and its extracts are widely used as nutrients for humans and animals, natural colorants in food and cosmetics and nutraceuticals and food additives for products of the pharmaceutical industry, BC protect against certain chronic diseases such as cancer, diabetes, cardiovascular diseases etc, [7]. This microalgae extracts have been attributed antidiabetic properties due to the induction of recovery of damaged pancreatic β-cells through its properties as antioxidants, anti-inflammatory and anti-apoptotic [8]. But there are no reports in case of stem cell differentiation to produce IPCs with phycoextract inclusion.

In this work, we with AP extract and BC inclusion in each three stages and throughout the differentiation process [2] and focused on the evaluation during the differentiation process of hADMSCs and hDPMSCs to IPCs.

Material and Methods

Extraction Process

Arthrospira Platensis LB-23 (from university of Texas, Austin) was cultivated in UTEX spirulina medium, the biomass obtained was crushed in a mortar with glass beads and adding methanol (10 ml/g) at 25°C and then centrifuged at 4°C at 10,000 rpm for 15 minutes. The supernatant was evaporated at 30°C in a Speedvac (Vacufuge-Eppendorf), then, the extract was solubilized in Phosphate-Buffered Saline (PBS) (1:10 v/v) and filtered with Whatman filters of 0.22 μg PES (Polyethersulfon, 28420282). The efficiency of extraction was calculated according to the following formula:

Analysis of AP Extract by TLC, UV/VIS AND HPLC

For qualitative analysis of BC, Thin Layer Chromatography (TLC) technique was used. Silica gel plates were used as a stationary phase, mounted on 20 x 10 cm glass and a mixture of Hexane: Acetone used at a proportion of 70:30 (v/v), the extract was analyzed together with the standard BC (C9750-5G Sigma-Aldrich), with the running time of 20 minutes [9]. The Rf was calculated with the following formula:

A spectrophotometric method was also used for quantification at a wavelength of 455 nm [10], by reading in triplicate and comparing with a standard curve constructed using commercially available BC (C9750-5G-SIGMA) in a plate reader (Eon BioTek). Finally, an HPLC analysis was performed for quantitative analysis. The standard of BC was diluted in acetone for standard curve generation and sample quantification, using a high-efficiency liquid chromatograph (Varian ProStar) with autosampler, a Luna column was used (LC column 3μm-Phenomex 00F-4162-E0), as a mobile phase a mixture of Hexane: Acetone 82:18 (v / v) was used. Additionally, a standard of astaxanthin was included using the same conditions.

Characterization of hMSCs

The hADMSCs were obtained from ATCC (PCS-500-011, lot 62098855), with hMSCs characterization certificate. The hDPMSCs were isolated from the third molar from a 22-year-old North American male patient, all procedures were carried out in accordance with the relevant institutional laws and guidelines and approved by the appropriate institutional committees. Informed consent was obtained for experimentation with human subjects. The dental piece was extracted by exodontia collected under the ethical criteria without reported pathologies. The procedures to isolate the stem cells of the dental pulp were those mentioned by Karamzadeh [11], modifying the enzymatic solution, the digestion of the dental pulp was carried out by adding 1ml of 0.025% trypsin, the obtained cells they underwent phenotypic characterization, according to International Society of Cell Therapy (ISCT).

Flow cytometry analysis was performed to identify membrane markers for hMSCs like CD44, CD73, CD90, and CD105 as well as the absence of hematopoietic markers using the analysis kit BD (562245). Multi-differentiation was also performed for the hDPMSCs to osteocytes, adipocytes, and chondrocytes following the specifications of the ISCT. For this purpose, cells were cultured for 24 days in specific differential media under standard culture conditions like for the case of osteoblasts, the Human Osteoblast Differentiation medium was used (Sigma Aldrich 417D). Later Von Kossa staining was used to observe calcium deposits, which was done by adding 500 μl of 5% silver nitrate (AgNO3) and subjected to UV radiation at 120 μJ/cm2 for 5 minutes. For the differentiation of adipocytes, the Human Adipocyte Differentiation medium (Sigma Aldrich 811D) was used. The staining with oily red was used to demonstrate the presence of fat vacuoles. After the culturing the cells with Chondrocyte Differentiation medium (Sigma Aldrich 411D), it was stained with a solution of 1% alcian blue. hDPMSCs without subjecting differentiation treatment, were subjected to the same stains, these being the controls.

Viability Assay

Both types of hMSCs were grown in D'MEM/F-12 (Sigma D8437) enriched with L-glutamine and 10% FBS (Fetal Bovine Serum) under standard culture conditions. They were placed in six-well culture plates, utilizing different concentrations of the AP extract and BC standard diluted with culture medium DMEM/F-12 free of FBS for 72 hours. The cells were exposed to the extract and to the standard at different concentrations (3, 1.5, 0.75, 0.375 and 0 μM) under standard culture conditions at 37°C, 95% relative humidity and 5% CO2.

A follow-up was made by microscopic observation to visualize if there was cellular damage in terms of morphological changes or cellular detachment produced by the AP extract an BC. Viability analysis was performed by cytometry, the cells were harvested using Trypsin/EDTA (ATCC PCS-999-003), and the cell pellet was resuspended in PBS. Then viability test was carried out by flow cytometry using the BD Cell Feasibility Kit viability kit (349383) and the BD Accuri C6 flow cytometer, using the Side Scatter (SCC) and Forward Scatter (FCC) graphics and the FL3-FL1 channel.

Differentiation of hMSCs into Insulin-Producing Cells

Differentiation of hADMSCs

The process of differentiation of the hADMSCs was carried out using the protocol by Chandra [12]. This process consisting of three stages: stage 1 (of hMSCs to meso-endodermal cell; stage 2 (of meso-endoderms to endocrine progenitors) and stage 3 (of endocrine progenitors to IPCs), during the experiment, AP extract was added to cultures in all the stages at a concentration of 1.5 μM, as well as a control of differentiation without AP extract and a control without differentiation protocol was maintained. In other experiments, AP extract was added at stage-1 only, stage-2 only and stage-3 only respectively. The same process was followed the BC standard was used.

Differentiation of hDPMSCs

The process of differentiation of the hDPMSCs was carried out by using the protocol established by our research group, which consisted of three stages. In the first stage, serum free D´MEM/F-12, 17.5 mM Glucose, Activin A 100 ng/mL and CHIR 3μM was used for five days at 37 °C and 5% CO2. On the fifth day the change was made to stage 2 which contained serum-free D´MEM/F-12, 17.5 mM glucose, FGF 4 ng/mL, IGF 50 ng/mL, Noggin 100 ng/mL, 1μM Dorsomorphin, 2 μM Retinoic Acid for five days. On the tenth day the change was made to stage 3 which contained serum-free DMEM F-12, 17.5 mM glucose, 10μM Forskolin, 3 μM Taurine, 10mM Nicotinamide, Dexamethasone 10 μM, Supplement B27 1% for five days. During the experiment, AP extract was added to culture at 1.5 μM, as well as a control of differentiation without AP extract and a control without undergoing the protocol differentiation was maintained. In other instances, AP extract was added at stage-1 only, stage-2 only and stage-3 only respectively. The same process was followed to use the standard of BC.

Analysis of Intracellular Insulin

Once the differentiation protocols were concluded, the cells were harvested with scrapper, then stained with anti-insulin antibodies Alexa Fluor 647 (565689) and analyzed in a Accuri C6 Flow Cytometer BD, permeabilizing the hDPMSCs and hADMSCs with methanol: acetone 1:1 (v/v) samples in the flow cytometer in channel FL-3 at 670 nm.

Gene Expression Study

Gene expression profiling was done to determine in vitro cellular responses of differentiation of hMSCs with and withouth β-carotene. At the end of each differentiation stage, cells were collected to carry out an RT-PCR analysis in order to look for MAFA, PDX1 and NKX6.1 genes transcripts, β-actin was used as a reference gene (Table 1).