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Progressive morphologic changes during generation and maturation of hHFMSC-derived erythroid cells in vitro. (a) Progressive morphologic changes from hematopoietic-like cells, erythroblasts to enucleated erythrocytes, and eventually matured erythrocytes are accompanied by significant increase of hemoglobin and decrease in size during their in vitro differentiation and maturation. Cells were stained with Wright-Giemsa dye. (b) Diameter decreased with time in culture. Data for each day represent diameters of cells. Enucleated cells decreased to less than half the original diameter on day 3 and were 3 times smaller than the macroblast on day 7. (c) Nuclear-to-cytoplasm ratio decreased with time in culture.
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Shortage of red blood cells (RBCs, erythrocytes) can have potentially life-threatening consequences for rare or unusual blood type patients with massive blood loss resulting from various conditions. Erythrocytes have been derived from human pluripotent stem cells (PSCs), but the risk of potential tumorigenicity cannot be ignored, and a majority of...
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The production of blood cells and their precursors from human pluripotent stem cells (hPSCs) in vitro has the potential to make a significant impact upon healthcare provision. We demonstrate that the forward programming of hPSCs through overexpression of GATA1, FLI1, and TAL1 leads to the production of a population of progenitors that can different...
Citations
... In our earlier research, we discovered that human hair follicle mesenchymal stem cells (hHFMSCs) derived from the bulge and papilla of hair follicles can be induced into iPSCs by the introduction of four pluripotent factors (Oct4, Sox2, c-Myc, and Klf4) [3] . Additionally, we found that introducing only OCT4 can reprogram hHFMSCs into intermediate cells (hHFMSCs OCT4 ), and these reprogrammed cells can be used to generate mature erythrocytes directly by stimulating them with speci c cytokines [4] . Furthermore, we observed an intriguing phenomenon in which pre-stimulation with low concentrations of FLT-3 and SCF cytokines resulted in the generation of suspended cell subsets capable of in vitro passage and expansion, as well as delaying differentiation. ...
... Furthermore, we observed an intriguing phenomenon in which pre-stimulation with low concentrations of FLT-3 and SCF cytokines resulted in the generation of suspended cell subsets capable of in vitro passage and expansion, as well as delaying differentiation. Upon sequential stimulation by a cascade of hematopoietic factors, the suspended cells underwent prompt differentiation into erythroid cells [4] . As a result, this process eliminates the need for sorting HSCs and maintaining their status. ...
... As a result, this process eliminates the need for sorting HSCs and maintaining their status. Signi cantly, a unique subset of suspended cells displaying distinct cellular morphology characteristics was identi ed, characterized by a small volume, round shape, and low adhesion, and subsequently designated as oating cells derived from hHFMSC OCT4 ( oating hHFMSCs OCT4 ) [4,5] . Nevertheless, the correlation between alterations in morphology and adhesion of reprogrammed cells and their capacity for self-renewal remained ambiguous. ...
Background: The challenge of expanding haematopoietic stem/progenitor cells (HSPCs) in vitro has limited the clinical application of this technology. Human hair follicle mesenchymal stem cells (hHFMSCs) can be reprogrammed to generate intermediate stem cells by introducing only OCT4 (hHFMSCsOCT4). Prestimulating these cells with a low concentration of the cytokines FLT3 and SCF leads to the generation of a distinct subset, named floating hHFMSCsOCT4, which can maintain self-renewal in vitro and be induced to undergo erythropoiesis, suggesting that these cells are promising seed cells for artificial haematopoiesis.
Methods: Floating cell subsets were isolated from adherent cell subsets using centrifugation. Cell adhesion was assessed through disassociation and adhesion assays. OCT4 expression levels were measured using immunofluorescence staining, RT-qPCR, and Western blotting. RNA sequencing and Gene Ontology (GO) enrichment analysis were then conducted to identify proliferation-related biological processes enriched by the upregulated differentially expressed genes (DEGs). Proliferative capacity was assessed using CCK-8 and colony formation assays. Cytoskeleton was observed through Wright‒Giemsa, Coomassie brilliant blue, and phalloidin staining. Expression of adherens junction (AJ) core members was confirmed through RT‒qPCR, Western blotting, and immunofluorescence staining before and after ZO-1 knockdown. A regulatory network was constructed to determine relationships among cytoskeleton, proliferation, and the AJ pathway. Student's t tests (GraphPad Prism 8.0.2) were used for group comparisons. The results were considered significant at P < 0.05.
Results: Excessive OCT4 expression weakens adhesion and causes floating hHFMSCsOCT4. These cells have moderate proliferation and undergo cytoskeleton remodeling, with increased contraction and aggregation of F-actin near the nucleus. The upregulation of ZO-1 can impact the actin, E-cadherin, and β-catenin genes, as well as the nuclear positioning of β-catenin, leading to variations in the cytoskeleton and cell cycle. Finally, a regulatory network revealed that the AJ pathway critically bridges cytoskeletal remodelling and haematopoiesis-related proliferation in a β-catenin-dependent manner.
Conclusions: Increasing OCT4 levels can remodel the cytoskeleton through the ZO-1-containing AJ pathway, potentially boosting the self-renewal ability of hHFMSCs to mimic HSPCs, suggesting a plausible mechanism for utilizing novel seed cells in artificial hematopoietic in vitro.
... Sun et al. group reported that intravenous injection of HF-MSCs to the rats with acute pancreatitis regenerated damaged pancreas and reduced IL-6 and TNF-α in the serum [21]. The pluripotent factor octamer-binding transcription factor 4 generated HF-MSCs can differentiate to enucleated adult-type erythrocytes, providing a new pathway for patient-specific transfusion [22]. Furthermore, engineered HF-MSCs released human insulin in a controlled manner that reversed hyperglycemia in mice with type 1 diabetes [23]. ...
Background
Alopecia areata (AA) is a common autoimmune hair loss disease with increasing incidence. Corticosteroids are the most widely used for hair loss treatment; however, long-term usage of hormonal drugs is associated with various side effects. Mesenchymal stem cells (MSCs) therapy has been studied extensively to curb autoimmune diseases without affecting immunity against diseases.
Methods
Hair follicle-derived MSCs (HF-MSCs) were harvested from the waste material of hair transplants, isolated and expanded. The therapeutic effect of HF-MSCs for AA treatment was investigated in vitro AA-like hair follicle organ model and in vivo C3H/HeJ AA mice model.
Results
AA-like hair follicle organ in vitro model was successfully established by pre-treatment of mouse vibrissa follicles by interferon-γ (IFN-γ). The AA-like symptoms were relieved when IFN-γ induced AA in vitro model was co-cultured with HF-MSC for 2 days. In addition, when skin grafted C3H/HeJ AA mice models were injected with 10⁶ HF-MSCs once a week for 3 weeks, the transcription profiling and immunofluorescence analysis depicted that HF-MSCs treatment significantly decreased mouse hair loss and reduced inflammation around HF both in vitro and in vivo.
Conclusions
This study provides a new therapeutic approach for alopecia areata based on HF-MSCs toward its future clinical application.
... MSCs are mesoderm-derived multifunctional stromal cells that have been identified in various tissues and organs, such as bone marrow, muscle, adipose tissue, pulp, placenta, hair follicles, brain periosteum, and umbilical cord [13][14][15]. Accumulating evidence has demonstrated that MSCs exhibit excellent therapeutic effects in treating various inflammatory conditions [16], autoimmune disorders [17], and degenerative diseases [18]. ...
Mesenchymal stem cells (MSCs) are considered to be a promising therapeutic material due to their capacities for self-renewal, multilineage differentiation, and immunomodulation and have attracted great attention in regenerative medicine. However, MSCs may lose their biological functions because of donor age or disease and environmental pressure before and after transplantation, which hinders the application of MSC-based therapy. As a major intracellular lysosome-dependent degradative process, autophagy plays a pivotal role in maintaining cellular homeostasis and withstanding environmental pressure and may become a potential therapeutic target for improving MSC functions. Recent studies have demonstrated that the regulation of autophagy is a promising approach for improving the biological properties of MSCs. More in-depth investigations about the role of autophagy in MSC biology are required to contribute to the clinical application of MSCs. In this review, we focus on the role of autophagy regulation by various physical and chemical factors on the biological functions of MSCs in vitro and in vivo, and provide some strategies for enhancing the therapeutic efficacy of MSCs.
... OCT4 (POU5F1), the core reprogramming factor, plays an important role in the maintenance of self-renewal and pluripotency of embryonic stem cells (ESCs). Therefore, the researchers transduced single factor OCT4 to hHFMSCs and stimulated with a series of hematopoietic cytokine stimulation to induce OCT4-reprogrammed human hair follicle mesenchymal stem cells (hHFMSCs OCT4 ) transdifferentiate towards the hematopoietic lineage [6]. After transduction, a population of small round floating cells with subtle expression of hematopoietic stem cell (HSC) marker CD45, gradually emerged from hHFMSCs OCT4 , and could transdifferentiate into mature enucleated RBCs when stimulated with a combination of hematopoietic cytokines [6], while the remaining cells formed negligible hematopoietic colony. ...
... Therefore, the researchers transduced single factor OCT4 to hHFMSCs and stimulated with a series of hematopoietic cytokine stimulation to induce OCT4-reprogrammed human hair follicle mesenchymal stem cells (hHFMSCs OCT4 ) transdifferentiate towards the hematopoietic lineage [6]. After transduction, a population of small round floating cells with subtle expression of hematopoietic stem cell (HSC) marker CD45, gradually emerged from hHFMSCs OCT4 , and could transdifferentiate into mature enucleated RBCs when stimulated with a combination of hematopoietic cytokines [6], while the remaining cells formed negligible hematopoietic colony. This prompted us to consider an association between this particular cell morphology and adhesion with possible erythropoiesis mechanisms, that is, low adhesion and round-like cell morphology conferring higher hematopoietic capacity to hHFMSCs OCT4 when treated with cytokines, thus promoting transduction of cellular signals and subsequently initiating the process of erythropoiesis. ...
... It was reported that floating hHFMSCs OCT4 treated with hematopoietic cytokines could directly transdifferentiate into enucleated RBCs expressing β-globin [6]. Here, we investigated the correlation between cell morphology, adhesion, pluripotency, and hematopoiesis to elucidate the mechanisms of erythropoiesis in OCT4reprogrammed hHFMSCs. ...
Background
Human hair follicle mesenchymal stem cells (hHFMSCs) isolated from hair follicles possess multilineage differentiation potential. OCT4 is a gene critically associated with pluripotency properties. The cell morphology and adhesion of hHFMSCs significantly changed after transduction of OCT4 and two subpopulations emerged, including adherent cells and floating cell. Floating cells cultured in hematopoietic induction medium and stimulated with erythropoetic growth factors could transdifferentiate into mature erythrocytes, whereas adherent cells formed negligible hematopoietic colonies. The aim of this study was to reveal the role of cell morphology and adhesion on erythropoiesis induced by OCT4 in hHFMSCs and to characterize the molecular mechanisms involved.
Methods
Floating cell was separated from adherent cell by centrifugation of the upper medium during cell culture. Cell size was observed through flow cytometry and cell adhesion was tested by disassociation and adhesion assays. RNA sequencing was performed to detect genome-wide transcriptomes and identify differentially expressed genes. GO enrichment analysis and KEGG pathway analysis were performed to analysis the functions and pathways enriched by differentially expressed genes. The expression of tight junction core members was verified by qPCR and Western blot. A regulatory network was constructed to figure out the relationship between cell adhesin, cytoskeleton, pluripotency, and hematopoiesis.
Results
The overexpression of OCT4 influenced the morphology and adhesion of hHFMSCs. Transcripts in floating cells and adherent cells are quite different. Data analysis showed that upregulated genes in floating cells were mainly related to pluripotency, germ layer development (including hematopoiesis lineage development), and downregulated genes were mainly related to cell adhesion, cell junctions, and the cytoskeleton. Most molecules of the tight junction (TJ) pathway were downregulated and molecular homeostasis of the TJ was disturbed, as CLDNs were disrupted, and JAMs and TJPs were upregulated. The dynamic expression of cell adhesion-related gene E-cadherin and cytoskeleton-related gene ACTN2 might cause different morphology and adhesion. Finally, a regulatory network centered to OCT4 was constructed, which elucidated that he TJ pathway critically bridges pluripotency and hematopoiesis in a TJP1-dependent way.
Conclusions
Regulations of cell morphology and adhesion via the TJ pathway conducted by OCT4 might modulate hematopoiesis in hHFMSCs, thus developing potential mechanism of erythropoiesis in vitro.
... Lentivirus vector pLV-EF1α-OCT4-IRES-EGFP and packaging plasmids expressing gag-pol, pVSVG, and rev genes were obtained from the Institute of Biochemistry and Cell Biology of Shanghai Life Science Research Institute, Chinese Academy of Science. Lentivirus production and transduction were performed according to our previous protocol (Liu et al., 2015). MDA-MB-231 cells and MCF7 cells were seeded on a 6-well plate and were infected with lentivirus expressing OCT4 in the presence of 5 mg/ml of polybrene for 24 h. ...
Background: Metastatic breast cancer is the major cause of death in breast cancer patients. Activation of epithelial-mesenchymal transition (EMT) induces migration and invasion of breast cancer cells (BCCs). OCT4 (POU5F1) is a key transcription factor for reprograming and plays an important role in self-renewal. Recent studies recovered OCT4 may correlate with cancer progression. However, it is no sufficient proofs to verify how OCT4 plays in metastasis of breast cancer. In this present study, we show the role of OCT4 in the migration and invasion of BCCs in vitro and metastasis in vivo.
Methods: PCR, Western Blot and Immunofluorescence staining were performed to determine to OCT4 expression in BCCs. Wound-healing assay and invasion assay were utilized to analyze the mobility of BCCs. Tumor metastasis was assessed with nude mice by subcutaneously injection. IHC assay was used to evaluate phosphorylated signal transducer and activator of transcription 3 (p-STAT3) expression in breast cancer tissues and normal breast tissues. To study whether OCT4 regulate EMT through STAT3 signal, we used shRNA to knockdown STAT3 gene expression in BCCs.
Results: OCT4 changed cell morphology of BCCs, decreased cell adhesion, and inhibited migration, invasion and metastatic ability of BCCs. In the meantime, overexpression of OCT4 activated STAT3 signaling and changed EMT-related protein expressions in BCCs. However, knockdown of STAT3 in BCCs with overexpression of OCT4 could facilitate EMT.
Conclusion: Our data demonstrate that OCT4 suppresses EMT in BCCs through activation of STAT3 signaling, which is a key mechanism in impeding BCCs migration and invasion. Collectively, these data suggest that elevating OCT4 expression may be an effective method for reducing the metastatic potential of BCCs, which could also contribute to developing new methods for diagnosis and new molecular target therapies in breast cancer metastasis.
... Although RBCs have been derived from human PSCs, the risk of potential tumorigenicity cannot be ignored, and a majority of these cells produced from PSCs express embryonic e-globins and fetal g-globins with little or no adult b-globin and remain nucleated 32 . Lu and colleagues reported a method to generate RBCs from human hair follicle MSCs (hHFMSCs) by enforcing OCT4 gene expression and cytokine stimulation 32 . The adult b-globin chain with a minimum level of the fetal g-globin chain was found in the cells generated from hHFMSCs. ...
Red blood cell (RBC) transfusion is a common therapeutic intervention, which is necessary for patients with emergency or hematological disorders to reduce morbidity and mortality. However, to date, blood available for transfusion is a limited resource, and the transfusion coverage system still depends on the volunteer-based collection system. The scarcity of blood supplies commonly develops because of local conditions that transiently affect collection. Moreover, donor-derived infectious disease transmission events also remain a risk. Thus, there is a huge demand for artificial blood. The production of cultured RBCs from stem cells is slowly emerging as a potential alternative to donor-derived red cell transfusion products. In this concise review, we summarize the recent in vitro expansion of RBCs from various stem cell sources, targeted therapy, prospects, and remaining challenges.
... Inducing erythrocyte production in vitro provides a model system for exploring the mechanisms of erythropoiesis. Previously, a population of small round oating cells with subtle expression of hematopoietic stem cell (HSC) marker CD45, gradually emerged from OCT4(POU5F1)-reprogrammed human hair follicle mesenchymal stem cells (hHFMSCs OCT4 ), and could transdifferentiate into mature enucleated RBCs when stimulated with a combination of hematopoietic cytokines (1). This prompted us to consider an association between this particular cell morphology and adhesion with possible erythropoiesis mechanisms, that is, low adhesion and round-like cell morphology conferring higher hematopoietic capacity to hHFMSCs OCT4 when treated with cytokines, thus promoting transduction of cellular signals and subsequently initiating the process of erythropoiesis. ...
... hHFMSCs are easily available and nonimmunogenic, making them a potential alternative stem cell source for patient-speci c applications. Transduction of the individual factor OCT4 allows hHFMSCs to transdifferentiate into RBCs under multiple hematopoietic induction conditions, providing an optional way to generate RBCs in vitro for transfusion (1). Evolutionary conservation analysis was performed to identify a relative subset of targets of OCT4 and other POU proteins that link the regulation of cell-cell adhesion to differentiation (25). ...
Background: Human hair follicle mesenchymal stem cells (hHFMSCs) isolated from hair follicles possess multilineage differentiation potential. OCT4 is a gene critically associated with pluripotency properties. The cell morphology and adhesion of hHFMSCs significantly changed after transduction of OCT4 and two subpopulations emerged, including adherent cells and floating cell. Floating cells cultured in hematopoietic induction medium and stimulated with erythropoetic growth factors could transdifferentiate into mature erythrocytes, whereas adherent cells formed negligible hematopoietic colonies. The aim of this study was to reveal the role of cell morphology and adhesion on erythropoiesis induced by OCT4 in hHFMSCs and to characterize the molecular mechanisms involved.
Methods: Floating cell were separated from adherent cell by centrifugation of the upper medium during cell culture. Cell size was observed through flow cytometry and cell adhesion was tested by disassociation and adhesion assays. RNA sequencing was performed to detect genome-wide transcriptomes and identify differentially expressed genes. GO enrichment analysis and KEGG pathway analysis were performed to analysis the functions and pathways enriched by differentially expressed genes. The expression of tight junction core members was verified by qPCR and Western blot.
Results: The overexpression of OCT4 influenced the morphology and adhesion of hHFMSCs. Transcripts in floating cells and adherent cells are quite different. Data analysis showed that upregulated genes in floating cells were mainly related to the pluripotency, germ layer development (including hematopoiesis lineage development), and downregulated genes were mainly related to cell adhesion, cell junctions and the cytoskeleton. Most molecules of the tight junction (TJ) pathway were downregulated and molecular homeostasis of the TJ was disturbed, as CLDNs were disrupted, and JAMs and TJPs were upregulated. The dynamic expression of cell adhesion-related gene E-cadherin and cytoskeleton-related gene ACTN2 might cause different morphology and adhesion. Finally, a regulatory network centered to OCT4 was constructed, which elucidated the TJ pathway critically bridges pluripotency and hematopoiesis in a TJP1-dependent way.
Conclusions: Regulations of cell morphology and adhesion via the TJ pathway conducted by OCT4 might modulate hematopoiesis in hHFMSCs, thus developing potential mechanism of erythropoiesis in vitro.
... OCT4 (POU5F1), the core reprogramming factor, plays an important role in the maintenance of self-renewal and pluripotency of embryonic stem cells (ESCs). Therefore, the researchers transduced single factor OCT4 to hHFMSCs and stimulated with a series of hematopoietic cytokine stimulation to induce OCT4reprogrammed human hair follicle mesenchymal stem cells (hHFMSCs OCT4 ) transdifferentiate towards the hematopoietic lineage (6). After transduction, a population of small round oating cells with subtle expression of hematopoietic stem cell (HSC) marker CD45, gradually emerged from hHFMSCs OCT4 , and could transdifferentiate into mature enucleated RBCs when stimulated with a combination of hematopoietic cytokines (6), while the remaining cells formed negligible hematopoietic colony. ...
... Therefore, the researchers transduced single factor OCT4 to hHFMSCs and stimulated with a series of hematopoietic cytokine stimulation to induce OCT4reprogrammed human hair follicle mesenchymal stem cells (hHFMSCs OCT4 ) transdifferentiate towards the hematopoietic lineage (6). After transduction, a population of small round oating cells with subtle expression of hematopoietic stem cell (HSC) marker CD45, gradually emerged from hHFMSCs OCT4 , and could transdifferentiate into mature enucleated RBCs when stimulated with a combination of hematopoietic cytokines (6), while the remaining cells formed negligible hematopoietic colony. This prompted us to consider an association between this particular cell morphology and adhesion with possible erythropoiesis mechanisms, that is, low adhesion and round-like cell morphology conferring higher hematopoietic capacity to hHFMSCs OCT4 when treated with cytokines, thus promoting transduction of cellular signals and subsequently initiating the process of erythropoiesis. ...
... It was reported that oating hHFMSCs OCT4 treated with hematopoietic cytokines could directly transdifferentiate into enucleated RBCs expressing β-globin (6). Here, we investigated the correlation between cell morphology, adhesion, pluripotency and hematopoiesis to elucidate the mechanisms of erythropoiesis in OCT4-reprogrammed hHFMSCs. ...
Background: Human hair follicle mesenchymal stem cells (hHFMSCs) isolated from hair follicles possess multilineage differentiation potential. OCT4 is a gene critically associated with pluripotency properties. The cell morphology and adhesion of hHFMSCs significantly changed after transduction of OCT4 and two subpopulations emerged, including adherent cells and floating cell. Floating cells cultured in hematopoietic induction medium and stimulated with erythropoetic growth factors could transdifferentiate into mature erythrocytes, whereas adherent cells formed negligible hematopoietic colonies. The aim of this study was to reveal the role of cell morphology and adhesion on erythropoiesis induced by OCT4 in hHFMSCs and to characterize the molecular mechanisms involved.
Methods: Floating cell were separated from adherent cell by centrifugation of the upper medium during cell culture. Cell size was observed through flow cytometry and cell adhesion was tested by disassociation and adhesion assays. RNA sequencing was performed to detect genome-wide transcriptomes and identify differentially expressed genes. GO enrichment analysis and KEGG pathway analysis were performed to analysis the functions and pathways enriched by differentially expressed genes. The expression of tight junction core members was verified by qPCR and Western blot. A regulatory network was constructed to figure out the relationship between cell adhesin, cytoskeleton, pluripotency and hematopoiesis.
Results: The overexpression of OCT4 influenced the morphology and adhesion of hHFMSCs. Transcripts in floating cells and adherent cells are quite different. Data analysis showed that upregulated genes in floating cells were mainly related to pluripotency, germ layer development (including hematopoiesis lineage development), and downregulated genes were mainly related to cell adhesion, cell junctions and the cytoskeleton. Most molecules of the tight junction (TJ) pathway were downregulated and molecular homeostasis of the TJ was disturbed, as CLDNs were disrupted, and JAMs and TJPs were upregulated. The dynamic expression of cell adhesion-related gene E-cadherin and cytoskeleton-related gene ACTN2 might cause different morphology and adhesion. Finally, a regulatory network centered to OCT4 was constructed, which elucidated that he TJ pathway critically bridges pluripotency and hematopoiesis in a TJP1-dependent way.
Conclusions: Regulations of cell morphology and adhesion via the TJ pathway conducted by OCT4 might modulate hematopoiesis in hHFMSCs, thus developing potential mechanism of erythropoiesis in vitro.
... A previous study showed that dermal papilla and dermal sheath cells generate hematopoietic colonies in vitro, and can contribute to multi-lineage hematopoietic reconstitution in vivo after transplantation into lethally irradiated recipient mice [3] . Recently, Liu et al [49] induced mature erythrocytes from hHF-MSCs by overexpressing OCT4 and hematopoietic cytokine exposure. This mature erythrocyte contained no nuclei, and expressed mainly the adult β-globin chain and rarely the fetal γ-globin chain. ...
Hair follicles are easily accessible skin appendages that protect against cold and potential injuries. Hair follicles contain various pools of stem cells, such as epithelial, melanocyte, and mesenchymal stem cells (MSCs) that continuously self-renew, differentiate, regulate hair growth, and maintain skin homeostasis. Recently, MSCs derived from the dermal papilla or dermal sheath of the human hair follicle have received attention because of their accessibility and broad differentiation potential. In this review, we describe the applications of human hair follicle-derived MSCs (hHF-MSCs) in tissue engineering and regenerative medicine. We have described protocols for isolating hHF-MSCs from human hair follicles and their culture condition in detail. We also summarize strategies for maintaining hHF-MSCs in a highly proliferative but undifferentiated state after repeated in vitro passages, including supplementation of growth factors, 3D suspension culture technology, and 3D aggregates of MSCs. In addition, we report the potential of hHF-MSCs in obtaining induced smooth muscle cells and tissue-engineered blood vessels, regenerated hair follicles, induced red blood cells, and induced pluripotent stem cells. In summary, the abundance, convenient accessibility, and broad differentiation potential make hHF-MSCs an ideal seed cell source of regenerative medical and cell therapy.
... Hair follicles are an easily accessible rich source of autologous stem cells, exhibiting tremendous advantages over other cell sources in various clinical applications. Indeed, the use of hair follicle mesenchymal stem cells (HF-MSCs) as a cell source for skin wound healing, hair follicle regeneration, nerve repair, cardiovascular tissue engineering, and gene therapy has shown remarkable success [26][27][28][29]. In a previous study, we successfully use transgenic HF-MSCs overexpressing the release-controlled insulin gene to reverse hyperglycemia and decrease mortality rates in streptozotocin-induced diabetic mice [30]. ...
Background:
PBX homeobox 1 (PBX1) is involved in the maintenance of the pluripotency of human embryonic and hematopoietic stem cells; however, the effects of PBX1 in the self-renewal and reprogramming of hair follicle mesenchymal stem cells (HF-MSCs) are unclear. The AKT/glycogen synthase kinase (GSK) 3β pathway regulates cell metabolism, proliferation, apoptosis, and reprogramming, and p16 and p21, which act downstream of this pathway, regulate cell proliferation, cell cycle, and apoptosis induced by reprogramming. Here, we aimed to elucidate the roles of PBX1 in regulating the proliferation and reprogramming of HF-MSCs.
Methods:
A lentiviral vector designed to carry the PBX1 sequence or PBX1 short hairpin RNA sequence was used to overexpress or knock down PBX1. The roles of PBX1 in proliferation and apoptosis were investigated by flow cytometry. Real-time polymerase chain reaction was performed to evaluate pluripotent gene expression. Dual-luciferase reporter assays were performed to examine the transcriptional activity of the NANOG promoter. Western blotting was performed to identify the molecules downstream of PBX1 involved in proliferation and reprogramming. Caspase3 activity was detected to assess HF-MSC reprogramming. The phosphatidylinositol 3-kinase/AKT inhibitor LY294002 was used to inhibit the phosphorylation and activity of AKT.
Results:
Overexpression of PBX1 in HF-MSCs increased the phosphorylation of AKT and nuclear translocation of β-catenin, resulting in the progression of the cell cycle from G0/G1 to S phase. Moreover, transfection with a combination of five transcription factors (SOMKP) in HF-MSCs enhanced the formation of alkaline phosphatase-stained colonies compared with that in HF-MSCs transfected with a combination of four transcription factors (SOMK). PBX1 upregulated Nanog transcription by activating the promoter and promoted the expression of endogenous SOX2 and OCT4. Furthermore, PBX1 expression activated the AKT/glycogen synthase kinase (GSK) 3β pathway and reduced apoptosis during the early stages of reprogramming. Inhibition of phospho-AKT or knockdown of PBX1 promoted mitochondrion-mediated apoptosis and reduced reprogramming efficiency.
Conclusions:
PBX1 enhanced HF-MSC proliferation, and HF-MSCs induced pluripotent stem cells (iPSC) generation by activating the AKT/GSK3β signaling pathway. During the reprogramming of HF-MSCs into HF-iPSCs, PBX1 activated the NANOG promoter, upregulated NANOG, and inhibited mitochondrion-mediated apoptosis via the AKT/GSK3β pathway during the early stages of reprogramming.
