Somatic Sources Research Articles

An optimized protocol for generating appendage-bearing skin organoids from human-induced pluripotent stem cells.

Organoid generation from pluripotent stem cells is a cutting-edge technique that has created new possibilities for modelling human organs in vitro, as well as opening avenues for regenerative medicine. Here, we present a protocol for generating skin organoids (SKOs) from human-induced pluripotent stem cells (hiPSCs) via direct embryoid body formation. This method provides a consistent start point for hiPSC differentiation, resulting in SKOs with complex skin architecture and appendages (e.g. hair follicles, sebaceous glands, etc.) across hiPSC lines from two different somatic sources.

Considerations for enhanced mesenchymal stromal/stem cell myogenic commitment invitro.

The generation of tissue from stem cells is an alluring concept as it holds a number of potential applications in clinical therapeutics and regenerative medicine. Mesenchymal stromal/stem cells (MSCs) can be isolated from a number of different somatic sources, and have the capacity to differentiate into adipogenic, osteogenic, chondrogenic, and myogenic lineages. Although the first three have been extensively investigated, there remains a paucity of literature on the latter. This review looks at the various strategies available invitro to enhance harvested MSC commitment and differentiation into the myogenic pathway. These include chemical inducers, myogenic-enhancing cell culture substrates, and mechanical and dynamic culturing conditions. Drawing on information from embryonic and postnatal myogenesis from somites, satellite, and myogenic progenitor cells, the mechanisms behind the chemical and mechanical induction strategies can be studied, and the sequential gene and signaling cascades can be used to monitor the progression of myogenic differentiation in the laboratory. Increased understanding of the stimuli and signaling mechanisms in the initial stages of MSC myogenic commitment will provide tools with which we can enhance their differentiation efficacy and advance the process to clinical translation.

Generation of Directly Reprogrammed Human Endothelial Cells.

Direct reprogramming provides a novel breakthrough for generating functional endothelial cells (ECs) without the need for intermediate stem or progenitor states, offering a promising resource for cardiovascular research and treatment. ETV2 is a key transcription factor that has been identified as a pioneering factor for specifying endothelial lineage. Achieving precise ETV2 induction is essential for effective endothelial reprogramming, and maintaining the reprogrammed cellular phenotype relies on a specific combination of growth factors and small molecules. Thus, we hereby provide a straightforward and comprehensive protocol for generating two distinct types of reprogrammed ECs (rECs) from human dermal fibroblasts (HDFs). Early rECs demonstrate a robust neovascularization property but lack the mature EC phenotype, while late rECs exhibit phenotypical similarity to human postnatal ECs and have a neovascularization capacity similarto early rECs. Both cell types can be derived from human somatic source cells, making them suitable for personalized disease investigations, drug discovery, and disease therapy.

The incidence of post cholecystectomy pain (PCP) syndrome at 12months following laparoscopic cholecystectomy: a prospective evaluation in 200 patients.

Post cholecystectomy pain syndrome can cause significant distress, impairs quality of life and exacerbations often result in emergency visits. Poorly controlled postoperative pain is a recognized cause of persistent postsurgical pain. Abdominal myofascial pain syndrome is an underdiagnosed cause of persistent pain in this cohort. The objective was to estimate the incidence of poorly controlled postoperative pain in the first 48 h after surgery and the likelihood of developing persistent pain at 12 months. The patients undergoing laparoscopic cholecystectomy at a tertiary unit were consented for participation in a prospective service evaluation. A telephone review was performed at three, six and twelve months after surgery. Incidence of poorly controlled pain in the first 48 h after surgery was assessed. Patients with persistent pain were referred to the pain clinic. Over a six-month period, 200 patients were assessed. Eleven patients were excluded (5.5 %). Twelve patients were lost to follow-up (6.6 %, 12/189). Patient satisfaction with acute postoperative pain management was low in 40 % (76/189). Poorly controlled postoperative pain was reported by 36 % (68/189) of patients. Incidence of persistent pain was 29 % (54/189) at 12 months post-surgery. Over half of patients with persistent pain (63 %, 34/54) reported poorly controlled postoperative pain. A somatic source was diagnosed in 54 % (29/54) with post cholecystectomy pain syndrome. Poorly controlled postoperative pain was reported by a third of patients. Persistent pain was present in 29 % at twelve months post-surgery. Abdominal myofascial pain syndrome should be considered as a differential diagnosis in post cholecystectomy pain syndrome.

신경정신분석의 이론적 기초 I

Psychoanalysis and neuroscience are the same in that both study the mind, although their methodologies are different. Neuropsychoanalysis, which makes integrative approaches to them, is needed for the mind studies. The paper attempts to explore the neuropsychoanalytic concept of drive by examining the drive-related theories of Freud, Lacan, Damasio, Panksepp, and Solms. According to Freud, drive arises from somatic sources and operates at the unconscious level. Affects, emotions, and feelings are based on drives, but they are felt and realized at the conscious levels. He focuses only on the psychological due to the intellectual limitations of his times but makes it clear that the sources of drive are somatic. Lacan refuses to attend to the physical source of drive. For Damasio, feelings arise primarily from the physical sources, and the feelings include background/primitive feelings, primary emotion feelings, and secondary emotion feelings. Feelings are fundamental to the mind and accompany emotions. Freud’s drive and Damasio’s feeling concepts are similar in that they indicate the basic energy of the mind arising from the somatic. Panksepp presents four major emotional systems on the ground of the sources of feelings within intrinsic brain functions. Solms follows Panksepp’s ideas. For both Damasio and Panksepp, feelings/emotional systems emerge from somatic sources, and the sources are a variety of neural circuits and neurochemicals. If we can fill with Damasio’s and Panksepp’s similar ideas of neural circuits and neurochemicals the somatic sources that Freud left blank, we can think of the neuropsychoanalytic concept of drive.

Establishment of iPSC (KRIBBi001-A) from CD34+ group O D-negative bone marrow blood.

Human induced pluripotent stem cells with indefinite propagation in vitro provide a potential donor source of cells including erythroid cells for human therapy. Since group O D-negative (RhD-) blood cells are considered as universal donors for transfusion, it is compelling to derive iPSC line from group O/RhD- sample as a new cellular source to generate universal RBCs. The resulting iPSC line derived from group O/RhD- somatic source showed typical features of pluripotent stem cells and could provide an unprecedented cellular tool to develop universal therapeutics for blood transfusion.

Abstract 330: Expression of Glycolytic Enzymes in Induced Pluripotent Stem Cells, Derived Megakaryocytes, and Endothelial Cells

Introduction: Induced pluripotent stem cells (iPSC) have a significant capacity for self-renewal and can differentiate into any somatic cell type, such as megakaryocytes (MK) and vascular endothelial cells (EC). We have previously shown that iPSC-derived MKs (IPSC-MK) upregulate the gene expression levels of irreversible glycolytic enzymes committing glucose to glycolysis in comparison to their parental iPSC lines (Vaidya et al., 2016). This pattern differed from past comparisons between somatic source cells and iPSCs (Chung et al., 2007; Varum et al., 2011; Zhang et al., 2011). To further corroborate our data, we extended our analysis to iPSC-derived ECs (iPSC-EC). Methods: We studied expression of glycolytic enzyme genes in 30 human iPSC lines and paired iPSC-EC and iPSC-MK lines using RNA-seq. iPSC-EC to iPSC expression ratios for 11 glycolytic enzymes were estimated using a multilevel mixed-effect model regression, and a significance threshold of p < 0.05/11 = 0.0045 was used. A bivariate statistical analysis was used to assess the correlation between iPSC-EC and iPSC-MK data. Results: Of the 11 glycolytic enzymes studied, 4 were highly expressed in iPSC-ECs (Log2(iPSC-EC/iPSC) ≥ 1) while 1 was highly expressed in iPSCs (Log2(iPSC/iPSC-EC) ≥ 1). Transcripts for functionally irreversible enzymes including ADPGK, PFKP, and PKLR displayed higher expression levels in iPSC-ECs compared to iPSCs. Similarly, 6 glycolytic genes demonstrated higher expression in iPSC-MKs, while 2 were more highly expressed in iPSCs. The functionally irreversible enzymes HK1, ADPGK, and PFK exhibited higher expression levels in iPSC-MKs. The ratios of gene expression (derived-lines:iPSCs) for all 37 genes in the iPSC-MK results correlated with those of the iPSC-EC results: Pearson’s r = 0.52, p = 0.001. Conclusion: Both iPSC-ECs and iPSC-MKs upregulate the expression of glycolytic enzymes compared to their parental iPSCs. In light of data showing that differentiated cells are more metabolically active and have more mitochondria than iPSCs (Chandra et al., 2020), these results suggest that differentiation of iPSCs is accompanied by an increase in both glycolytic and TCA cycle activities, with glycolytic generation of pyruvate used for mitochondrial production of ATP.

Human Induced Pluripotent Stem Cells Derived from a Cardiac Somatic Source: Insights for an In-Vitro Cardiomyocyte Platform.

Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) has revolutionized the complex scientific field of disease modelling and personalized therapy. Cardiac differentiation of human iPSCs into cardiomyocytes (hiPSC-CMs) has been used in a wide range of healthy and disease models by deriving CMs from different somatic cells. Unfortunately, hiPSC-CMs have to be improved because existing protocols are not completely able to obtain mature CMs recapitulating physiological properties of human adult cardiac cells. Therefore, improvements and advances able to standardize differentiation conditions are needed. Lately, evidences of an epigenetic memory retained by the somatic cells used for deriving hiPSC-CMs has led to evaluation of different somatic sources in order to obtain more mature hiPSC-derived CMs.

Addressing Variability and Heterogeneity of Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Induced pluripotent stem cells (iPSCs) offer great promise in the areas of disease modeling, basic research, drug development, and regenerative medicine. Much of their value comes from the fact that they can be used to create otherwise inaccessible cell types, such as cardiomyocytes, which are genetically matched to a patient or any other individual of interest. A consistent issue plaguing the iPSC platform, however, involves excessive variability exhibited in the differentiated products. This includes discrepancies in genetic, epigenetic, and transcriptional features, cell signalling, the cell types produced from cardiac differentiation, and cardiomyocyte functionality. These properties can result from both the somatic source cells and environmental conditions related to the derivation and handling of these cells. Understanding the potential sources of variability, along with determining which factors are most relevant to a given application, are essential in advancing iPSC-based technologies.

Porcine somatic cell nuclear transfer donor sources affect transplant success: A meta-analysis

Herd boars, male domestic pigs used for stud, are economically important, and somatic cell nuclear transfer (SCNT) is a promising technology to expand herd boar yields. However, live births are dictated by donor cell source, and fetal donors may offer more advantages than adult donors. A meta-analysis was conducted to better understand how donor sources affect SCNT outcomes. Of the 1,431 records viewed, 10 were selected for review. Blastocyst formation rates, successful pregnancies, and live births were assessed to measure efficacy. SCNT blastocyst formation differed between adult and fetal donors among the studies. SCNT pigs had more malformed fetuses as well, which negatively affected the post-birth mortality. Organs of porcine fetuses are limited by deficiencies of maternal nutrient and growth hormones, which compromise post-birth adaptations. SCNT pregnancy success is neither determined by donor source nor by live births. Live births are also tied to donor age. Embryos from fetal donors are more frequently healthy likely due to less differentiation and less reprogramming of reconstructed embryos. Adult donors in contrast have more cell differentiation and as such accumulate more mutations and damage. This may reduce reconstructed embryo viability. Finally, SCNT efficiency may be improved with more in vitro passages, but more work is required to validate this concept.

CXCL12/CXCR4 Signaling Enhances Human PSC-Derived Hematopoietic ProgenitorFunction and Overcomes Early InVivo Transplantation Failure.

SummaryHuman pluripotent stem cells (hPSCs) generate hematopoietic progenitor cells (HPCs) but fail to engraft xenograft models used to detect adult/somatic hematopoietic stem cells (HSCs) from donors. Recent progress to derive hPSC-derived HSCs has relied on cell-autonomous forced expression of transcription factors; however, the relationship of bone marrow to transplanted cells remains unknown. Here, we quantified a failure of hPSC-HPCs to survive even 24 hr post transplantation. Across several hPSC-HPC differentiation methodologies, we identified the lack of CXCR4 expression and function. Ectopic CXCR4 conferred CXCL12 ligand-dependent signaling of hPSC-HPCs in biochemical assays and increased migration/chemotaxis, hematopoietic progenitor capacity, and survival and proliferation following in vivo transplantation. This was accompanied by a transcriptional shift of hPSC-HPCs toward somatic/adult sources, but this approach failed to produce long-term HSC xenograft reconstitution. Our results reveal that networks involving CXCR4 should be targeted to generate putative HSCs with in vivo function from hPSCs.

Prospective Audit of a Pathway for In-Patient Pain Management of Chronic Abdominal Pain: A Novel and Cost-Effective Strategy.

Unexplained abdominal pain is a common cause of hospital admission and utilizes significant resource. Current in-patient pain management of acute exacerbation of chronic abdominal pain is primarily directed at pharmacological and psychological management strategies in this group of complex patients. We adopted a novel approach that proved to be both clinically effective and cost-effective. Adult patients admitted to a surgical ward with acute exacerbation of chronic abdominal pain referred to in-patient pain management were prospectively audited over a two-year period at a single tertiary centre. Management strategy focused on a somatic source as the predominant pain generator. Patients were offered ultrasound-guided trigger point injection with steroids within 48 hours of referral and were discharged when pain control was achieved. Subsequent care by the pain physician included targeted treatment of somatic component (repeated trigger point injection with steroids or pulsed radiofrequency treatment of trigger points). We audited 43 patients referred to the inpatient pain management service over a two-year period. Four patients refused to undergo the diagnostic trigger point injection. Three patients with active visceral disease had a transient response to the injection. Thirty-six patients were diagnosed with abdominal myofascial pain syndrome, and two-thirds of these patients were discharged home within 36 hours of the intervention. Abdominal myofascial pain syndrome is a poorly recognized cause of chronic abdominal pain, especially in patients with a past history of visceral inflammation. The novel strategy resulted in a significant reduction in opioid consumption, length of stay, and readmission rate.

Woman, body, earth and spirit: Journeys of descent through myth, embodiment and movement practice

Abstract In this article I describe some personal journeys, inwards through the deepest layers of the psyche, to discover embodied understanding of what I call the deep feminine, or the feminine ground of being. I explore some myths and ancient stories of the Goddess that reflect the journey of healing and transformation through psychological descent, and can guide us through such ordeals. As C. G. Jung discovered, myths and archetypes are rooted in the instinctual body; as a somatic movement practitioner and transpersonal and body-oriented psychotherapist, my own journeys led me to intuitive appreciations of the somatic sources of mythic journeys that have particular relevance to contemporary women seeking healing/wholeness and spiritual renewal. The myth of Inanna, the oldest known myth of the Goddess’s descent to the Underworld, affirms the truth that our healing and spiritual journey takes us through a process of ‘endarkenment’, in order to reach wholeness and discover the light of spirit within. The Goddess Lilith also represents the ‘dark’ side of the feminine, which we need to balance and reconcile with our upperworldly nature. La Loba, Wild Woman, shows us a path towards rebirth and revival of our soul and spirit. These ancient and timeless tales offer guidance and reflection for contemporary women, and also men exploring the feminine within their own psyches.

Integrated Genomic Analysis of Diverse Induced Pluripotent Stem Cells from the Progenitor Cell Biology Consortium.

SummaryThe rigorous characterization of distinct induced pluripotent stem cells (iPSC) derived from multiple reprogramming technologies, somatic sources, and donors is required to understand potential sources of variability and downstream potential. To achieve this goal, the Progenitor Cell Biology Consortium performed comprehensive experimental and genomic analyses of 58 iPSC from ten laboratories generated using a variety of reprogramming genes, vectors, and cells. Associated global molecular characterization studies identified functionally informative correlations in gene expression, DNA methylation, and/or copy-number variation among key developmental and oncogenic regulators as a result of donor, sex, line stability, reprogramming technology, and cell of origin. Furthermore, X-chromosome inactivation in PSC produced highly correlated differences in teratoma-lineage staining and regulator expression upon differentiation. All experimental results, and raw, processed, and metadata from these analyses, including powerful tools, are interactively accessible from a new online portal at https://www.synapse.org to serve as a reusable resource for the stem cell community.

Capturing the ephemeral human pluripotent state.

During human development, pluripotency is present only in early stages of development. This ephemeral cell potential can be captured in vitro by obtaining pluripotent stem cells (PSC) with self-renewal properties, the human embryonic stem cells (hESC). However, diverse studies suggest the existence of a plethora of human PSC (hPSC) that can be derived from both embryonic and somatic sources, depending on defined culture conditions, their spatial origin, and the genetic engineering used for reprogramming. This review will focus on hPSC, covering the conventional primed hESC, naïve-like hPSC that resemble the ground-state of development, region-selective PSC, and human induced PSC (hiPSC). We will analyze differences and similarities in their differentiation potential as well as in the molecular circuitry of pluripotency. Finally, we describe the need for human feeder cells to derive and maintain hPSC, because they could emulate the interaction of in vivo pluripotent cells with extraembryonic structures that support development. Developmental Dynamics 245:762-773, 2016. © 2016 Wiley Periodicals, Inc.

Reprogramming and transdifferentiation for cardiovascular development and regenerative medicine: where do we stand?

Heart disease remains a leading cause of mortality and a major worldwide healthcare burden. Recent advances in stem cell biology have made it feasible to derive large quantities of cardiomyocytes for disease modeling, drug development, and regenerative medicine. The discoveries of reprogramming and transdifferentiation as novel biological processes have significantly contributed to this paradigm. This review surveys the means by which reprogramming and transdifferentiation can be employed to generate induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and induced cardiomyocytes (iCMs). The application of these patient-specific cardiomyocytes for both in vitro disease modeling and in vivo therapies for various cardiovascular diseases will also be discussed. We propose that, with additional refinement, human disease-specific cardiomyocytes will allow us to significantly advance the understanding of cardiovascular disease mechanisms and accelerate the development of novel therapeutic options.

PTEN inactivation by germline/somatic c.950_953delTACT mutation in patients with Lhermitte–Duclos disease manifesting progressive phenotypes

Lhermitte-Duclos disease (LDD), a neurological manifestation of Cowden syndrome (CS), is a rare and benign cerebellar disorder, featured by dysplastic cerebellar ganglion cells which replace granular and Purkinje cells. Phosphatase and Tensin Homolog (PTEN) is confirmed as the susceptibility gene for CS which represents the most complex features and is not easily recognizable. We reported two index patients with LDD diagnosed either in an isolated form or coexist with CS. These two patients displayed progressive though comparable phenotypes and were found to carry an identical PTEN c.950_953delTACT mutation in either germline or somatic sources of DNA, respectively. Negative or moderate expression levels of PTEN were validated by immunohistochemistry in the corresponding patients' affected tissues. This study has revealed a novel pathogenicity locus to LDD/CS as a candidate for early molecular diagnosis. In addition, the differential PTEN mutation status with corresponding LDD phenotypes suggests a potential correlation between germline or somatic mutation and coexisting LDD/CS or isolated LDD, respectively. Furthermore, our data could lend some reference to the underlying molecular mechanism of LDD pathogenesis in the future.

Disease-Causing Mitochondrial Heteroplasmy Segregated Within Induced Pluripotent Stem Cell Clones Derived from a Patient with MELAS

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.

Nuclear Reprogramming with c-Myc Potentiates Glycolytic Capacity of Derived Induced Pluripotent Stem Cells

Reprogramming strategies influence the differentiation capacity of derived induced pluripotent stem (iPS) cells. Removal of the reprogramming factor c-Myc reduces tumorigenic incidence and increases cardiogenic potential of iPS cells. c-Myc is a regulator of energy metabolism, yet the impact on metabolic reprogramming underlying pluripotent induction is unknown. Here, mitochondrial and metabolic interrogation of iPS cells derived with (4F) and without (3F) c-Myc demonstrated that nuclear reprogramming consistently reverted mitochondria to embryonic-like immature structures. Metabolomic profiling segregated derived iPS cells from the parental somatic source based on the attained pluripotency-associated glycolytic phenotype and discriminated between 3F versus 4F clones based upon glycolytic intermediates. Real-time flux analysis demonstrated a greater glycolytic capacity in 4F iPS cells, in the setting of equivalent oxidative capacity to 3F iPS cells. Thus, inclusion of c-Myc potentiates the pluripotent glycolytic behavior of derived iPS cells, supporting c-Myc-free reprogramming as a strategy to facilitate oxidative metabolism-dependent lineage engagement.

In Vivo Generation of Neural Tumors from Neoplastic Pluripotent Stem Cells Models Early Human Pediatric Brain Tumor Formation

Recent studies have identified gene signatures in malignant tumors that are associated with human embryonic stem cells, suggesting a molecular relationship between aggressive cancers and pluripotency. Here, we characterize neural precursors (NPs) derived from transformed human embryonic stem cells (N-t-hESCs) that exhibit neoplastic features of human brain tumors. NPs derived from t-hESCs have enhanced cell proliferation and an inability to mature toward the astrocytic lineage, compared with progeny derived from normal human embryonic stem cells (N-hESCs) independent of adherent or neurosphere outgrowth. Intracranial transplantation of NPs derived from N-t-hESCs and N-hESCs into NOD SCID mice revealed development of neuroectoderm tumors exclusively from the N-t-hESCs NPs and not from normal N-hESCs. These tumors infiltrated the ventricles and the cerebellum of recipient mice and displayed morphological, phenotypic, and molecular features associated with classic medulloblastoma including retention of a pluripotent signature. Importantly, N-t-hESCs did not exhibit cytogenetic changes associated with medulloblastoma, suggesting that aberrant cellular and molecular properties precede the acquisition of karyotypic changes thus underscoring the value of this model system of human medulloblastoma. Our study demonstrates that NPs from a starting population of neoplastic human pluripotent parent cells possess brain tumor-initiating cell capacity, thereby providing a model system to investigate initiation and progression of primitive human neural cancers that are difficult to assess using somatic sources.