Building Muscle: The Role of Insulin-Like Growth Factor Binding Protein-6 and Insulin-Like Growth Factors in the Differentiation of Placental Mesenchymal Stem Cells into Skeletal Muscle

Human placenta is a readily available source for isolation of adult mesenchymal stem cell (MSC), for potential use in regenerative therapies. MSC fate is influenced by the microenvironment in vivo, and insulin-like growth factors (IGFs) are critical components of the stem cell niche, as they regulate proliferation and differentiation into different lineages including bone, fat, and skeletal muscle. Insulin-like growth factor binding protein-6 (IGFBP-6), relative to other IGFBPs, has high affinity for IGF2 and is believed to be the main modulator of IGF-2 function. However, the role of IGFBP-6 in muscle development has not been clearly defined. In this study, we investigated the role of IGFBP-6 in different stages of muscle commitment and differentiation using human mesenchymal stem cells derived from the placenta (PMSCs). Our central hypothesis is that IGFBP-6 regulates the maintenance of multipotency in PMSCs and also promotes PMSC differentiation into muscle via intracellular and extracellular interactions in both IGF-dependent and independent mechanisms. We used immunoblotting, immunocytochemistry, ELISA, and ALDH-activity to evaluate IGFBP-6 effects on PMSC muscle differentiation. We showed that PMSCs are capable of differentiating into muscle cells when exposed to muscle-specific differentiation medium characterized by the decrease of pluripotency-associated markers (OCT4 and SOX2) and the gain of expression of muscle markers Pax3/7, MyoD, Myogenin, and Myosin heavy chain in a time-dependent manner and eventually forming multi-nucleated fibers. Extracellular supplementation with IGFBP-6 during culture increased muscle differentiation markers levels in early stages. The opposite effects were observed when IGFBP-6 was silenced and was rescued by increasing IGFBP-6. We also showed that IGFBP-6 had impact on muscle differentiation in both IGF-dependent and -independent mechanisms. IGF-1 and IGF-2 had different effects on muscle differentiation with IGF-1 promoting multipotency and early commitment to muscle, whereas IGF-2 promoting muscle differentiation. Muscle differentiation required activation of both AKT and MAPK pathways. Interestingly, we demonstrated that IGFBP-6 could compensate for IGF-2 loss and help enhance the muscle differentiation process by triggering predominantly the MAPK pathway independent of activating either IGF-1R or insulin receptor (IR). These findings indicate complex interactions between IGFBP-6 and IGFs in PMSC differentiation into skeletal muscle. The most prominent effects were observed early in the differentiation process, before muscle lineage commitment. This knowledge on how myogenesis can be manipulated using IGFs and IGFBP-6 will aid in the development of improved muscle regeneration therapies using stem cells from human placenta

THE ROLE OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-6 IN THE DIFFERENTIATION OF PLACENTAL MESENCHYMAL STEM CELLS INTO SKELETAL MUSCLE

Insulin-like growth factor binding protein-6 (IGFBP-6), a component of the stem cell niche involved in the differentiation of skeletal muscle myogenesis, is expressed in developing muscle cells and is the main regulator of IGF-II. In this study, I investigated the role of IGFBP-6 in the commitment of skeletal muscle derivation from placenta mesenchymal stem cells (PMSCs). I hypothesized that IGFBP-6 inhibits the temporal maintenance of PMSCs and promotes the differentiation of PMSCs into muscle via both extracellular and intracellular mechanisms. PMSCs can differentiate into muscle cells expressing the muscle markers Pax3/7, MyoD, and Myogenin with the formation of multi-nucleated fibers. Under differentiation conditions, silencing IGFBP-6 increased Pax3/7 and decreased OCT4 levels. In contrast, under non-differentiation conditions, there was a significant increase in Pax3/7 levels at day 7 with intracellular and extracellular increase of IGFBP-6, similarly silencing IGFBP-6 under non-differentiation conditions significantly increased Pax3/7 at 24 hours and decreased OCT4 levels over time same as in differentiation conditions. I concluded that increasing lGFBP-6 promotes PMSCs differentiation with more prominent effects at the beginning of the differentiation process, while silencing IGFBP-6 has more dramatic effects on PMSCs. Knowledge of the effects of IGFBP-6 on muscle differentiation will help improve strategies for skeletal muscle regeneration therapies using stem cells

Different Effects of Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor-2 on Myogenic Differentiation of Human Mesenchymal Stem Cells

Insulin-like growth factors (IGFs) are critical components of the stem cell niche, as they regulate proliferation and differentiation of stem cells into different lineages, including skeletal muscle. We have previously reported that insulin-like growth factor binding protein-6 (IGFBP-6), which has high affinity for IGF-2, alters the differentiation process of placental mesenchymal stem cells (PMSCs) into skeletal muscle. In this study, we determined the roles of IGF-1 and IGF-2 and their interactions with IGFBP-6. We showed that IGF-1 increased IGFBP-6 levels within 24 hours but decreased after 3 days, while IGF-2 maintained higher levels of IGFBP-6 throughout myogenesis. IGF-1 increased IGFBP-6 in the early phase as a requirement for muscle commitment. In contrast, IGF-2 enhanced muscle differentiation as shown by the expression of muscle differentiation markers MyoD, MyoG, and MHC. IGF-1 and IGF-2 had different effects on muscle differentiation with IGF-1 promoting early commitment to muscle and IGF-2 promoting complete muscle differentiation. We also showed that PMSCs acquired increasing capacity to synthesize IGF-2 during muscle differentiation, and the capacity increased as the differentiation progressed suggesting an autocrine and/or paracrine effect. Additionally, we demonstrated that IGFBP-6 could enhance the muscle differentiation process in the absence of IGF-2

Insulin-Like Growth Factor Binding Protein-6 Promotes the Differentiation of Placental Mesenchymal Stem Cells into Skeletal Muscle Independent of Insulin-Like Growth Factor Receptor-1 and Insulin Receptor.

As mesenchymal stem cells (MSCs) are being investigated for regenerative therapies to be used in the clinic, delineating the roles of the IGF system in MSC growth and differentiation

Insulin-Like Growth Factor Binding Protein-6 Alters Skeletal Muscle Differentiation of Human Mesenchymal Stem Cells

Insulin-like growth factor binding protein-6 (IGFBP-6), the main regulator of insulin-like growth factor-2 (IGF-2), is a component of the stem cell niche in developing muscle cells. However, its role in muscle development has not been clearly defined. In this study, we investigated the role of IGFBP-6 in muscle commitment and differentiation of human mesenchymal stem cells derived from the placenta. We showed that placental mesenchymal stem cells (PMSCs) have the ability to differentiate into muscle cells when exposed to a specific culture medium by expressing muscle markers Pax3/7, MyoD, myogenin, and myosin heavy chain in a stage-dependent manner with the ultimate formation of multinucleated fibers and losing pluripotency-associated markers, OCT4 and SOX2. The addition of IGFBP-6 significantly increased pluripotency-associated markers as well as muscle differentiation markers at earlier time points, but the latter decreased with time. On the other hand, silencing IGFBP-6 decreased both pluripotent and differentiation markers at early time points. The levels of these markers increased as IGFBP-6 levels were restored. These findings indicate that IGFBP-6 influences MSC pluripotency and myogenic differentiation, with more prominent effects observed at the beginning of the differentiation process before muscle commitment

The Roles of Insulin-Like Growth Factors in Mesenchymal Stem Cell Niche

Many tissues contain adult mesenchymal stem cells (MSCs), which may be used in tissue regeneration therapies. However, the MSC availability in most tissues is limited which demands expansion in vitro following isolation. Like many developing cells, the state of MSCs is affected by the surrounding microenvironment, and mimicking this natural microenvironment that supports multipotent or differentiated state in vivo is essential to understand for the successful use of MSC in regenerative therapies. Many researchers are, therefore, optimizing cell culture conditions in vitro by altering growth factors, extracellular matrices, chemicals, oxygen tension, and surrounding pH to enhance stem cells self-renewal or differentiation. Insulin-like growth factors (IGFs) system has been demonstrated to play an important role in stem cell biology to either promote proliferation and self-renewal or enhance differentiation onset and outcome, depending on the cell culture conditions. In this review, we will describe the importance of IGFs, IGF-1 and IGF-2, in development and in the MSC niche and how they affect the pluripotency or differentiation towards multiple lineages of the three germ layers

Protective potential of mesenchymal stem cells against COVID-19 during pregnancy

SARS-CoV-2 causes COVID-19. COVID-19 has led to severe clinical illnesses and an unprecedented death toll. The virus induces immune inflammatory responses specifically cytokine storm in lungs. Several published reports indicated that pregnant females are less likely to develop severe symptoms compared with non-pregnant. Putative protective role of maternal blood circulating fetal mesenchymal stem cells (MSCs) has emerged and have been put forward as an explanation to alleviated symptoms. MSCs with immune-modulatory, anti-inflammatory and anti-viral roles, hold great potential for the treatment of COVID-19. MSCs could be an alternative to treat infections resulting from the SARS-CoV-2 and potential future outbreaks. This review focuses on the MSCs putative protective roles against COVID-19 in pregnant females

HLA-based banking of induced pluripotent stem cells in Saudi Arabia

Abstract Background Human iPSCs' derivation and use in clinical studies are transforming medicine. Yet, there is a high cost and long waiting time associated with autologous iPS-based cellular therapy, and the genetic engineering of hypo-immunogenic iPS cell lines is hampered with numerous hurdles. Therefore, it is increasingly interesting to create cell stocks based on HLA haplotype distribution in a given population. This study aimed to assess the potential of HLA-based iPS banking for the Saudi population. Methods In this study, we interrogated the HLA database of the Saudi Stem Cell Donor Registry (SSCDR), containing high-resolution HLA genotype data from 64,315 registered Saudi donors at the time of analysis. This database was considered to be a representative sample of the Saudi population. The most frequent HLA haplotypes in the Saudi population were determined, and an in-house developed iterative algorithm was used to identify their HLA matching percentages in the SSCDR database and cumulative coverage. Subsequently, to develop a clinically relevant protocol for iPSCs generation, and to illustrate the applicability of the concept of HLA-based banking for cell therapy purposes, the first HLA-based iPS cell line in Saudi Arabia was generated. Clinically relevant methods were employed to generate the two iPS clones from a homozygous donor for the most prevalent HLA haplotype in the Saudi population. The generated lines were then assessed for pluripotency markers, and their ability to differentiate into all three germ layers, beating cardiomyocytes, and neural progenitors was examined. Additionally, the genetic stability of the HLA-iPS cell lines was verified by comparing the mutational burden in the clones and the original blood sample, using whole-genome sequencing. The standards set by the American College of Medical Genetics and Genomics (ACMG) were used to determine the clinical significance of identified variants. Results The analysis revealed that the establishment of only 13 iPSC lines would match 30% of the Saudi population, 39 lines would attain 50% coverage, and 596 lines would be necessary for over 90% coverage. The proof-of-concept HLA-iPSCs, which cover 6.1% of the Saudi population, successfully demonstrated pluripotency and the ability to differentiate into various cell types including beating cardiomyocytes and neuronal progenitors. The comprehensive genetic analysis corroborated that all identified variants in the derived iPSCs were inherently present in the original donor sample and were classified as benign according to the standards set by the ACMG. Conclusions Our study sets a road map for introducing iPS-based cell therapy in the Kingdom of Saudi Arabia. It underscores the pragmatic approach of HLA-based iPSC banking which circumvents the limitations of autologous iPS-based cellular therapies. The successful generation and validation of iPSC lines based on the most prevalent HLA haplotype in the Saudi population signify a promising step toward broadening the accessibility and applicability of stem cell therapies and regenerative medicine in Saudi Arabia

Generation of myoglobin (MB)-knockout human embryonic stem cell (hESC) line (KAIMRCe002-A-1S) using CRISPR/Cas9 technology

Myoglobin (MB) is a cytoplasmic hemoprotein that is predominantly expressed in the heart and oxidative myofibers of skeletal muscle. It has been demonstrated that MB binds to oxygen and promotes its diffusion for energy production in the mitochondria. Recently, MB was found to be expressed in different forms of malignant tumors and cancer cell lines. Further studies using gene disruption technology will enhance the understanding of MB’s role in human cardiovascular biology and cancers. Here, we describe the generation of a homozygous MB knockout in human embryonic stem cells (hESC-MB−/−) via CRISPR/Cas9 to study MB function in human biology and diseases