Source Of Pluripotent Cells Research Articles

KLF2 regulates stemness of human mesenchymal stem cells by targeting FGFR3

ABSTRACT Mesenchymal stem cells (MSCs) are an attractive source of pluripotent cells for regenerative therapy; however, maintaining stemness and self-renewal of MSCs during expansion ex vivo is challenging. For future clinical applications, it is essential to define the roles and signaling pathways that regulate the fate of MSCs. Based on our earlier finding that Krüppel-like factor 2 (KLF2) participates in maintaining stemness in MSCs, we examined further the role of this factor in intrinsic signaling pathways. Using a chromatin immunoprecipitation (ChIP)-sequence assay, we found that the FGFR3 gene is a KLF2 binding site. Knockdown of FGFR3 significantly decreased the levels of key pluripotency factors, enhanced the expression of differentiation-related genes and down-regulated colony formation of human bone marrow MSCs (hBMSCs). Using alizarin red S and oil red O staining, we found that knockdown of FGFR3 inhibited the osteogenic and adipogenic ability of MSCs under conditions of differentiation. The ChIP-qPCR assay confirmed that KLF2 interacts with the promoter regions of FGFR3. Our findings suggest that KLF2 promotes hBMSC stemness by direct regulation of FGFR. Our findings may contribute to enhanced MSC stemness by genetic modification of stemness-related genes.

BESC from cloned embryos do not retain transcriptomic or epigenetic memory from somatic donor cells.

Epigenetic reprogramming after mammalian somatic cell nuclear transfer is often incomplete, resulting in low efficiency of cloning. However, gene expression and histone modification analysis indicated high similarities in transcriptome and epigenomes of bovine embryonic stem cells from in vitro fertilized and somatic cell nuclear transfer embryos. Embryonic stem cells (ESC) indefinitely maintain the pluripotent state of the blastocyst epiblast. Stem cells are invaluable for studying development and lineage commitment, and in livestock, they constitute a useful tool for genomic improvement and in vitro breeding programs. Although these cells have been recently derived from bovine blastocysts, a detailed characterization of their molecular state is lacking. Here, we apply cutting-edge technologies to analyze the transcriptomic and epigenomic landscape of bovine ESC (bESC) obtained from in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. bESC were efficiently derived from SCNT and IVF embryos and expressed pluripotency markers while retaining genome stability. Transcriptome analysis revealed that only 46 genes were differentially expressed between IVF- and SCNT-derived bESC, which did not reflect significant deviation in cellular function. Interrogating histone 3 lysine 4 trimethylation, histone 3 lysine 9 trimethylation, and histone 3 lysine 27 trimethylation with cleavage under targets and tagmentation, we found that the epigenomes of both bESC groups were virtually indistinguishable. Minor epigenetic differences were randomly distributed throughout the genome and were not associated with differentially expressed or developmentally important genes. Finally, the categorization of genomic regions according to their combined histone mark signal demonstrated that all bESC shared the same epigenomic signatures, especially at gene promoters. Overall, we conclude that bESC derived from SCNT and IVF embryos are transcriptomically and epigenetically analogous, allowing for the production of an unlimited source of pluripotent cells from high genetic merit organisms without resorting to transgene-based techniques.

ABO gene editing for the conversion of blood type A to universal type O in Rhnull donor-derived human-induced pluripotent stem cells.

The limited availability of red cells with extremely rare blood group phenotypes is one of the global challenges in transfusion medicine that has prompted the search for alternative self‐renewable pluripotent cell sources for the in vitro generation of red cells with rare blood group types. One such phenotype is the Rhnull, which lacks all the Rh antigens on the red cell membrane and represents one of the rarest blood types in the world with only a few active blood donors available worldwide. Rhnull red cells are critical for the transfusion of immunized patients carrying the same phenotype, besides its utility in the diagnosis of Rh alloimmunization when a high‐prevalence Rh specificity is suspected in a patient or a pregnant woman. In both scenarios, the potential use of human‐induced pluripotent stem cell (hiPSC)‐derived Rhnull red cells is also dependent on ABO compatibility. Here, we present a CRISPR/Cas9‐mediated ABO gene edition strategy for the conversion of blood type A to universal type O, which we have applied to an Rhnull donor‐derived hiPSC line, originally carrying blood group A. This work provides a paradigmatic example of an approach potentially applicable to other hiPSC lines derived from rare blood donors not carrying blood type O.

Botanicals and Oral Stem Cell Mediated Regeneration: A Paradigm Shift from Artificial to Biological Replacement.

Stem cells are a well-known autologous pluripotent cell source, having excellent potential to develop into specialized cells, such as brain, skin, and bone marrow cells. The oral cavity is reported to be a rich source of multiple types of oral stem cells, including the dental pulp, mucosal soft tissues, periodontal ligament, and apical papilla. Oral stem cells were useful for both the regeneration of soft tissue components in the dental pulp and mineralized structure regeneration, such as bone or dentin, and can be a viable substitute for traditionally used bone marrow stem cells. In recent years, several studies have reported that plant extracts or compounds promoted the proliferation, differentiation, and survival of different oral stem cells. This review is carried out by following the PRISMA guidelines and focusing mainly on the effects of bioactive compounds on oral stem cell-mediated dental, bone, and neural regeneration. It is observed that in recent years studies were mainly focused on the utilization of oral stem cell-mediated regeneration of bone or dental mesenchymal cells, however, the utility of bioactive compounds on oral stem cell-mediated regeneration requires additional assessment beyond in vitro and in vivo studies, and requires more randomized clinical trials and case studies.

Abstract 12102: TNNI3-K183del Mutation in Dilated Cardiomyopathy Patient-Specific iPSC-Derived Cardiomyocytes Affects Structural Proteins and Ion Channels, Leading to Impair the Function of Cardiomyocytes

Background: One of the causes for Dilated cardiomyopathy (DCM) are thought to be genetic defect. One mutation, Lys183 del mutation of the TNNI3, is suggested to be associated with left ventricular systolic dysfunction and transition from hypertrophic cardiomyopathy to DCM. However, the precise disease mechanism is not fully understood. Because cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CMs) possess many of the same characteristics as primary human CMs and can be generated in unlimited quantities from pluripotent cell sources, they are thought to be a promising research tools to model genetic cardiomyopathies. Therefore, we hypothesized that iPS-CMs with mutations in TNNI3 is useful for elucidating the pathological mechanism of DCM and examined the phenotype of iPS-CMs with mutations in TNNI3. Methods and Results: We generated iPSCs from the patient with Lys183 del mutation of the TNNI3, and simultaneously induced iPS-CMs. By immunofluorescence stain analysis, we identified well-formed parallel arrays of repeating sarcomeres in myofibrils from WT iPS-CMs. On the other hand, iPS-CMs with Lys183 del mutation of the TNNI3 had fewer myofibrils and abnormal, irregular sarcomeres. In addition, sarcomere length was shorter in iPS-CMs with mutation in TNNI3 than in WT iPS-CMs. By gene expression analysis, we identified that iPS-CMs with mutation in TNNI3 had lower expression of some molecules including structural proteins or ion channels than WT iPS-CM. Cell motion analysis revealed that WT iPS-CMs were synchronously beating in multi wells, whereas iPS-CMs with mutation in TNNI3 were not. In addition, contraction velocity (CV) and relaxation velocity (RV) were significantly lower in iPS-CMs with mutation in TNNI3 (CV; 4.98±0.05 μm/sec, RV; 4.01±0.01 μm/sec) compared with WT iPS-CM (CV; 14.25±0.03 μm/sec, RV; 7.04±0.03 μm/sec). Moreover, significant decrease in contraction deformation distance and relaxation deformation distance were observed in iPS-CMs with mutation in TNNI3 (0.96±0.004 μm, 0.91±0.01 μm, respectively), compared to WT iPS-CMs (2.56±0.004 μm, 2.18±0.008 μm, respectively). Conclusion: iPS-CMs with Lys183 del mutation of the TNNI3 showed the morphological and functional phenotypes of DCM at a cellular level.

Transcriptional, Protein-Level and Functional Profiling of Human Fetal Liver (FL)-Derived Hematopoietic Stem Cells (HSCs) at Single Cell Resolution

Intro: The complex and tightly regulated process of human hematopoietic development culminates in the production of hematopoietic stem cells (HSCs), which subsequently acquire functional competence and undergo expansion in the fetal liver (FL). The establishment of a high-resolution molecular signature of FL HSCs provides insights into HSC biology with potential utility in the purification and expansion of engraftable HSCs ex vivo and the generation of HSCs from pluripotent cell sources. To profile HSCs at this developmental stage, we performed CITE-Seq, a technique that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies to simultaneously map transcriptional and protein-level expression in individual cells. To connect expression profiles with functional engraftment, we have coupled this with transplantation assays in immunocompromised mice. Methods: In these studies, three populations of human FL cells were used: CD34- cells, CD34+ cells and CD34+ cells furtherenrichedby expression of GPI-80, a marker tightly linked to engraftment potential, to explicitly identify HSCs capable of long-term engraftment. These populations were stained with a panel of oligo-tagged antibodies, processed via the 10X Genomics platform, and sequenced (26,407 total cells). Results: Transplantation experiments using the same sorted fractions that were assayed by CITE-seq revealed superior engraftment potential of the GPI-80+ fraction, and thus enrichment for bona fide HSCs at the functional level. This functional signature coincided with enrichment for known HSC markers such as ITGA6 (CD49f), PROCR (EPCR), CD164, MLLT3, HLF, CLEC9A and HMGA2 at the transcriptional level. As such, by profiling >7000 GPI-80+ cells, we have achieved unprecedented resolution of the engraftable HSC compartment within the FL. Combined analysis of all captured FL fractions accurately recapitulated the hematopoietic landscape of the FL at this developmental stage, representing the expected hematopoietic lineages and cell types. To gain further insight into FL HSCs, we next focused on the CD34+ HSC/progenitor compartment where we tracked cluster dynamics upon functional HSC enrichment between the CD34+ bulk and GPI-80+ sample. We noted a prominent (4-fold) increase in a cluster marked by enrichment for genes including RGCC, LMNA, VIM, ID1 and ID3 as well as components of the AHR pathway, suggesting that this expression profile strongly correlates with engraftment potential. Notably, LMNA is expressed in postnatal HSCs and its expression has been shown to decrease upon aging (Grigoryan et al., 2018). In line with this, we also found that LMNA is more highly expressed in our prenatal FL HSPCs compared to postnatal HSPCs. This data suggests a potential role for LMNA in endowing FL HSCs with their superior engraftment potential compared to postnatal HSCs. To complement our transcriptomic characterization of engraftment potential, we also collected cell surface marker expression data based on sequencing of a series of antibody-derived tags (ADTs). This additional layer of information uncovered expression patterns that weren't readily apparent based on mRNA expression data and inspired us to use this ADT information to gate out populations of interest via in silico sorting. This enabled us to compare the transcriptional profiles associated with well-described HSC enrichment signatures to assess whether they represent equivalent cell populations. We compared CD34+CD90+CD49f+ (~Notta et al., 2011) vs CD34+CD133+GPI-80+ (~Sumide et al., 2018) vs CD34+EPCR+ (~Subramaniam et al., 2019) in silico sorted cells and found a strong overlap in enriched genes, suggesting that the transcriptomic signature corresponding to engraftment potential identified in this work is not exclusive to GPI-80+ sorted cells but represents engraftable HSCs beyond this enrichment strategy. Conclusion: We have achieved unprecedented resolution of the engraftable HSC compartment in the human FL. Combining transcriptional profiling of the engraftment potential of FL HSCs with cell surface level expression data provides an in-depth characterization of this unique and developmentally relevant HSC source. This data will be valuable in optimizing the purification and expansion of engraftable HSCs ex vivo as well as in guiding the in vitro generation of HSCs from pluripotent cell sources. Disclosures No relevant conflicts of interest to declare.

3139 – TRANSCRIPTIONAL, PROTEIN-LEVEL AND FUNCTIONAL PROFILING OF HUMAN FETAL LIVER-DERIVED HEMATOPOIETIC STEM CELLS AT SINGLE CELL RESOLUTION

The complex and tightly regulated process of human hematopoietic development culminates in the production of hematopoietic stem cells (HSCs), which subsequently undergo expansion in the fetal liver (FL). The establishment of a high-resolution molecular signature of FL-HSCs would provide insights into HSC biology with potential utility in the purification and expansion of engraftable HSCs ex vivo and the generation of HSCs from pluripotent cell sources. To profile HSCs at this developmental stage, we performed CITE-Seq, a technique that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies. To connect expression profiles with functional engraftment, we have coupled this with transplantation assays in immunocompromised mice. In these studies, three populations of human FL cells were used: CD34- cells, CD34+ cells and CD34+ cells further enriched by expression of GPI-80, a marker tightly linked to engraftment potential, to acquire additional resolution of HSCs capable of long-term engraftment. These populations were stained with a panel of oligo-tagged antibodies, processed via the 10X platform, and sequenced (26.582 total cells). Simultaneous transplantation experiments of these three populations revealed superior engraftment potential of the CD34+GPI-80+ fraction, and thus enrichment for bona fide HSCs at the functional level. This coincided with enrichment for known HSC markers such as ITGA6 (CD49f), PROCR (EPCR), CD164, MLLT3, HLF and HMGA2 at the transcriptional level. As such, by profiling profiling >7000 GPI-80+CD34+ cells, we have achieved unprecedented resolution of the engraftable HSC compartment within the FL. Studying the transcriptional profile in combination with protein level expression of key HSC-related genes, will allow for an accurate characterization of these cells and provide insights into the biology behind their remarkable engraftment potential.

Cell-Based Therapeutic Approaches for Cystic Fibrosis.

Cystic Fibrosis (CF) is a chronic autosomal recessive disease caused by defects in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Cystic Fibrosis affects multiple organs but progressive remodeling of the airways, mucus accumulation, and chronic inflammation in the lung, result in lung disease as the major cause of morbidity and mortality. While advances in management of CF symptoms have increased the life expectancy of this devastating disease, and there is tremendous excitement about the potential of new agents targeting the CFTR molecule itself, there is still no curative treatment. With the recent advances in the identification of endogenous airway progenitor cells and in directed differentiation of pluripotent cell sources, cell-based therapeutic approaches for CF have become a plausible treatment method with the potential to ultimately cure the disease. In this review, we highlight the current state of cell therapy in the CF field focusing on the relevant autologous and allogeneic cell populations under investigation and the challenges associated with their use. In addition, we present advances in induced pluripotent stem (iPS) cell approaches and emerging new genetic engineering methods, which have the capacity to overcome the current limitations hindering cell therapy approaches.

Review on the Root, Stem and Leaf Initiations in Plants

The root and shoot apical meristem serve as sources of pluripotent cells and provide new cells for repetitive organ initiation, they are the major meristematic regions on which plant development take place. New meristems are incessantly formed as plants produce new branches or lateral roots thus making the understanding of meristem function central to how plants can establish different growth types, ranging from tiny herbs to huge trees. The sizes and numbers of meristems that are initiated during advanced development control the size and number of fruits and the generation of seeds. The development of a lateral root from a limited number of cells requires compactly coordinated asymmetric cell divisions to generate cell diversity and tissue patterns which characteristically involves the specification of founder cells, followed by a number of cellular changes until the cells divide and give rise to unequally sized daughter cells. Leaf development exemplifies the dynamic nature and flexibility of plant development in response to internal and external cues which is evidenced in the fact that two plants, even if genetically identical, do not look the same, two leaves on the same plant are different, and the final shape of a leaf is not predetermined when it starts to form. Leaves evolved from lateral branches following the acquisition of determinate growth and a flat structure, thus the specification of organ initiation involves a complex network of genetic, hormonal and mechanical factors which has been discussed in this review.

The scanning gradient Fourier transform (SGFT) method for assessing sarcomere organization and alignment

Recent advances in stem cell biology have allowed researchers to efficiently produce large numbers of cardiomyocytes from various pluripotent cell sources. Unfortunately, these cells exhibit properties that are characteristics of immature cardiomyocytes such as poor sarcomere organization, limited calcium handling, and reduced cell size and alignment. Specifically, the actin–myosin motor proteins that form sarcomeres within these cardiomyocytes fail to produce large, highly ordered repeating structures that are distinctive for adult myocytes. Instead, these cells produce heterogeneous sarcomeres that vary in thickness, alignment, and level of organization. Additionally, a large number of cardiomyopathies have been linked to mutations in genes encoding for sarcomeric proteins, resulting in disrupted sarcomere organization. This research focuses on a series of algorithms that provide a quantitative analysis technique to characterize the alignment and organization of sarcomere structures within aggregates and single cardiomyocytes. The scanning gradient Fourier transform (SGFT) method incorporates gradient analysis along with fast Fourier transforms to determine regions of sarcomere organization within individual and a population of cells, yielding a quantitative method of determining sarcomere organization and alignment at the sub-cellular scale. The utility of the SGFT technique is also demonstrated for additional applications, such as breast cancer collagen microstructure and neural rosette patterning.

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Highly efficient induced pluripotent stem cell reprogramming of cryopreserved lymphoblastoid cell lines.

Tissue culture based in-vitro experimental modeling of human inherited disorders provides insight into the cellular and molecular mechanisms involved and the underlying genetic component influencing the disease phenotype. The breakthrough development of induced pluripotent stem cell (iPSC) technology represents a quantum leap in experimental modeling of human diseases, providing investigators with a self-renewing and thus unlimited source of pluripotent cells for targeted differentiation into functionally relevant disease specific tissue/cell types. The existing rich bio-resource of Epstein-Barr virus (EBV) immortalized lymphoblastoid cell line (LCL) repositories generated from a wide array of patients in genetic and epidemiological studies worldwide, many of them with extensive genotypic, genomic and phenotypic data already existing, provides a great opportunity to reprogram iPSCs from any of these LCL donors in the context of their own genetic identity for disease modeling and disease gene identification. However, due to the low reprogramming efficiency and poor success rate of LCL to iPSC reprogramming, these LCL resources remain severely underused for this purpose. Here, we detailed step-by-step instructions to perform our highly efficient LCL-to-iPSC reprogramming protocol using EBNA1/OriP episomal plasmids encoding pluripotency transcription factors (i.e., OCT3/4, SOX2, KLF4, L-MYC, and LIN28), mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate (> 200 reprogrammed iPSC lines) using this protocol.

Activation-Induced Cytidine Deaminase Regulates Fibroblast Growth Factor/Extracellular Signal-Regulated Kinases Signaling to Achieve the Naïve Pluripotent State During Reprogramming.

Induced pluripotent stem cells (iPSCs) derived by in vitro reprogramming of somatic cells retain the capacity to self‐renew and to differentiate into many cell types. Pluripotency encompasses multiple states, with naïve iPSCs considered as ground state, possessing high levels of self‐renewal capacity and maximum potential without lineage restriction. We showed previously that activation‐induced cytidine deaminase (AICDA) facilitates stabilization of pluripotency during reprogramming. Here, we report that Acida −/− iPSCs, even when successfully reprogrammed, fail to achieve the naïve pluripotent state and remain primed for differentiation because of a failure to suppress fibroblast growth factor (FGF)/extracellular signal‐regulated kinases (ERK) signaling. Although the mutant cells display marked genomic hypermethylation, suppression of FGF/ERK signaling by AICDA is independent of deaminase activity. Thus, our study identifies AICDA as a novel regulator of naïve pluripotency through its activity on FGF/ERK signaling. stem cells 2019;37:1003–1017

Human embryonic stem cells: Distinct molecular personalities and applications in regenerative medicine.

The field of stem cell biology is exciting because it provides researchers and clinicians with seemingly unlimited applications for treating many human diseases. Stem cells are a renewable source of pluripotent cells that can differentiate into nearly all human cell types. In this article we focus particularly on human embryonic stem (hES) cells, derived from the inner cell mass of the blastocyst and cultured for expansion while remaining undifferentiated, to explore their unique molecular personalities and clinical applications. The aim of this literature review is to reflect the interest in hES cells and to provide a resource for researchers and clinicians interested in the molecular characteristics of such cells. Clin. Anat. 32:354–360, 2019. © 2018 The Authors. Clinical Anatomy published by Wiley Periodicals, Inc. on behalf of American Association of Clinical Anatomists.

Sheep embryonic stem-like cells engrafted into sheep femoral condyle osteochondral defects: 4-year follow-up

BackgroundArticular cartilage lacks a regenerative response. Embryonic stem cells (ESCs) are a source of pluripotent cells for cartilage regeneration. Their use, however, is associated with a risk of teratoma development, which depends on multiple factors including the number of engrafted cells and their degree of histocompatibility with recipients, the immunosuppression of the host and the site of transplantation.Colonies of sheep embryonic stem-like (ES-like) cells from in vitro-produced embryos, positive for stage-specific embryonic antigens (SSEAs), alkaline phosphatase (ALP), Oct 4, Nanog, Sox 2 and Stat 3 gene expression, and forming embryoid bodies, were pooled in groups of two-three, embedded in fibrin glue and engrafted into osteochondral defects in the left medial femoral condyles of 3 allogeneic ewes (ES). Empty defects (ED) and defects filled with cell-free glue (G) in the condyles of the controlateral stifle joint served as controls. After euthanasia at 4 years post-engraftment, the regenerated tissue was evaluated by macroscopic, histological and immunohistochemical (collagen type II) examinations and fluorescent in situ hybridization (FISH) assay to prove the ES-like cells origin of the regenerated tissue.ResultsNo teratoma occurred in any of the ES samples. No statistically significant macroscopic or histological differences were observed among the 3 treatment groups. FISH was positive in all the 3 ES samples.ConclusionsThis in vivo preclinical study allowed a long-term evaluation of the occurrence of teratoma in non-immunosuppressed allogeneic adult sheep engrafted with allogeneic ES-like cells, supporting the safe and reliable application of ES cells in the clinic.

Induced Pluripotent Stem Cells in Disease Modeling and Gene Identification.

Experimental modeling of human inherited disorders provides insight into the cellular and molecular mechanisms involved, and the underlying genetic component influencing, the disease phenotype. The breakthrough development of induced pluripotent stem cell (iPSC) technology represents a quantum leap in experimental modeling of human diseases, providing investigators with a self-renewing and, thus, unlimited source of pluripotent cells for targeted differentiation. In principle, the entire range of cell types found in the human body can be interrogated using an iPSC approach. Therefore, iPSC technology, and the increasingly refined abilities to differentiate iPSCs into disease-relevant target cells, has far-reaching implications for understanding disease pathophysiology, identifying disease-causing genes, and developing more precise therapeutics, including advances in regenerative medicine. In this chapter, we discuss the technological perspectives and recent developments in the application of patient-derived iPSC lines for human disease modeling and disease gene identification.

Translational Advances in the Management of Acute Spinal Cord Injury

Translational Advances in the Management of Acute Spinal Cord Injury

Xeno-Transplantation of macro-encapsulated islets and Pluripotent Stem Cell-Derived Pancreatic Progenitors without Immunosuppression.

Islet transplantation effectively treats diabetes but relies on immune suppression and is practically limited by the number of cadaveric islets available. An alternative cellular source is insulin-producing cells derived from pluripotent cell sources. Three animal cohorts were used in the current study to evaluate whether an oxygen-providing macro-encapsulation device, 'βAIR', could function in conjunction with human embryonic stem cells (hESCs) and their derivatives. The first cohort received macro-encapsulated undifferentiated hESCs, a second cohort received hESCs differentiated to a pancreatic progenitor state with limited endocrine differentiation. A reference cohort received human islets. Macro-encapsulation devices were implanted subcutaneously and monitored for up to 4 months. Undifferentiated pluripotent stem cells did not form teratoma but underwent cell death following implantation. Human C-peptide (hC- peptide) was detectable in host serum one week after implantation for both other cohorts. hC-peptide levels decreasing over time but remained detectable up to the end of the study. Key factors associated with mature endocrine cells were observed in grafts recovered from cohorts containing islets and hESC-derivatives including C-peptide, insulin, glucagon and urocortin 3. We conclude that the 'βAIR' macroencapsulation device is compatible with both human islets and pluripotent derivatives, but has a limited capability of sustaining undifferentiated pluripotent cells.

Subperiosteal Minimally Invasive Aesthetic Ridge Augmentation Technique (SMART): A New Standard for Bone Reconstruction of the Jaws.

Traditional guided bone regeneration techniques include flap mobilization and placement of a bone graft, often with the use of space-maintaining devices and cell-occlusive membranes. This approach is associated with frequent complications that negatively affect the outcome of the augmentation and the peri-implant soft tissue esthetics. Although current tunneling techniques have focused on periodontal soft tissue applications, earlier publications described their use for horizontal augmentation of mandibular posterior edentulous ridges in full-denture patients. More recently, the use of recombinant human platelet-derived growth factor (rhPDGF-BB) was tested with different bone matrices to treat maxillary anterior edentulous spans. The present case series reports the use of a subperiosteal minimally invasive aesthetic ridge augmentation technique (SMART) to treat 60 single and multiple edentulous, dentate, and implant sites on 21 patients and five treatment categories with a follow-up period ranging from 4 to 30 months. The technique includes the use of a laparoscopic approach to deliver a growth factor/xenograft combination into a subperiosteal pouch. No flap elevation, cell-occlusive membranes, space-maintaining devices, or decortication procedures were used. The results from this case series demonstrated predictable and consistent bone regeneration. The average gain in ridge width for all treatment categories was 5.11 mm (SD 0.76 mm), which compares favorably with previously published reports. Morbidity and complication rates were consistently reduced as well. Human histology results show xenograft particles surrounded by newly formed bone. The role of the periosteum as a source of pluripotent cells in growth factor–mediated bone regeneration is discussed.

Dedifferentiated Adipocytes Improve Heart Function Post-Myocardial Infarction

Background:The development of pluripotent stem cell-based strategies for regenerative medicine offers hope of one day having a limitless source of therapeutic cells for the repairing of damaged cardiac tissue resulting from myocardial infarction (MI). Dedifferentiated adipose cells (DFAT) have recently gained attention in this regard for their in vitro and in vivo ability to express vascular and cardiac-specific markers. To date, there is limited knowledge with respect to their capacity to improve cardiac function and contractility when introduced into the myocardium after the onset of MI. To that end, this study investigated the in vivo potential of intramyocardially injected mouse DFAT (mDFAT) cells to differentiate towards cardiovascular lineages and enhance cardiac function in a murine model of MI. Methods: Mature adipocytes were harvested from GFP-transgenic mice and dedifferentiated in vitro to produce pluripotent mDFAT cells. Mice that underwent LAD ligation surgery were injected intramyocardially with a dose of therapeutic mDFAT cells (n=6) shortly after the confirmation of successful MI. Results: Consistent with previous findings by the authors, we observed an immunofluorescent stain pattern in the infarct area of mDFAT cells adjacent to or co-localized with Troponin T, Cx-43, CD31, isolectin B4 and α-SMA up to eight weeks after cell transplantation. Furthermore, echocardiography and hemodynamic catheterization measurements indicated an overall improvement in global cardiac function, including ejection fraction, fractional shortening and contractility. Conclusions: This study is the first to report that the transplantation of mDFAT cells into a murine model of MI can preserve cardiac function up to eight weeks after the onset of infarction, suggesting a potential role for DFAT cells as a therapeutic pluripotent cell source in cardiovascular tissue repair.