Source Of Cells For Autologous Transplantation Research Articles

Induced Pluripotent Stem Cells and Genome-Editing Tools in Determining Gene Function and Therapy for Inherited Retinal Disorders.

Inherited retinal disorders (IRDs) affect millions of people worldwide and are a major cause of irreversible blindness. Therapies based on drugs, gene augmentation or transplantation approaches have been widely investigated and proposed. Among gene therapies for retinal degenerative diseases, the fast-evolving genome-editing CRISPR/Cas technology has emerged as a new potential treatment. The CRISPR/Cas system has been developed as a powerful genome-editing tool in ophthalmic studies and has been applied not only to gain proof of principle for gene therapies in vivo, but has also been extensively used in basic research to model diseases-in-a-dish. Indeed, the CRISPR/Cas technology has been exploited to genetically modify human induced pluripotent stem cells (iPSCs) to model retinal disorders in vitro, to test in vitro drugs and therapies and to provide a cell source for autologous transplantation. In this review, we will focus on the technological advances in iPSC-based cellular reprogramming and gene editing technologies to create human in vitro models that accurately recapitulate IRD mechanisms towards the development of treatments for retinal degenerative diseases.

Application of Autogenous Urine-Derived Stem Cell Sheet Enhances Rotator Cuff Healing in a Canine Model

Background: A repaired rotator cuff (RC) often heals with interposed scar tissue, making repairs prone to failure. Urine-derived stem cells (USCs), with robust proliferation ability and multilineage differentiation, can be isolated from urine, avoiding invasive and painful surgical procedures for harvesting the cells. These advantages make it a novel cell source for autologous transplantation to enhance RC healing. Hypothesis: Implantation of an autogenous USC sheet to the injury site will enhance RC healing. Study Design: Controlled laboratory study. Methods: USCs isolated from urine were cultured using ascorbic acid and transforming growth factor β3 to form a cell sheet. Sixteen male mature beagles underwent bilateral shoulder surgery. The right shoulder underwent infraspinatus tendon (IT) insertion detachment and repair only, and the other was subjected to IT insertion detachment and repair, followed by autogenous USC sheet implantation. Among the animals, 3 received a Dil (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)- labeled USC sheet implant in the right shoulder and were sacrificed at postoperative 6 weeks for cell tracking. The other animals were sacrificed at postoperative 12 weeks, and the IT-humerus complexes were harvested for gross observation, micro–computed tomography evaluation and histological analysis (n = 5), and mechanical testing (n = 8). Additionally, 13 unpaired canine cadaveric shoulders were included as native controls. Results: Micro–computed tomography analysis showed that the USC sheet group had a significant increase in bone volume/total volume and trabecular thickness at the RC healing site when compared with the control group (P < .05 for all). Histologically, the Dil-labeled USC sheet was still visible at the RC healing site, which suggested that the implanted USCs remained viable at postoperative 6 weeks. Meanwhile, the healing interface in the USC sheet group regenerated significantly more enthesis-like tissue than did that of the control group (P < .05). Additionally, the healing interface in the USC sheet group presented a larger fibrocartilage area, more proteoglycan deposition, and higher collagen birefringence than did that of the control group (P < .05 for all). Biomechanically, the USC sheet group showed significantly higher failure load and stiffness versus the control group (P < .05 for all). Conclusion: A USC sheet was able to enhance RC healing in a canine model. Clinical Relevance: The findings of the study showed that USC sheet implantation could serve as a practical application for RC healing.

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis.

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, an important number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow (BM), supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPC). To overcome the limit of BM harvest/HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex vivo expansion of HSPC coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1- defective patients were transduced with a clinically-optimized lentiviral vector expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NOD scid gamma common chain (NSG) recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPC coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by BM fibrosis.

The collagen scaffold supports hiPSC-derived NSC growth and restricts hiPSC.

The human induced pluripotent stem cells (hiPSC) are one of the promising candidates as patient specific cell source for autologous transplantation or modeling of diseases. The collagen (Col) scaffolds have been shown suitable to create in vitro biomimetic microenvironment for human neural stem cells, but their ability to accommodate stem cells at different stages of neural differentiation has not been verified yet. In this paper we compare lineage related hiPSC during neural differentiation for their ability to colonize Col scaffold. We have also focused on modification of collagen physicochemical properties with improved mechanical and thermal stability, without loss of its biological activity. The hiPSC expressing markers of pluripotency (OCT4, SOX2, NANOG) after neural commitment are NESTIN, GFAP, PDGFR alpha, beta- TUBULIN III, MAP-2, DCX, GalC positive. We have shown, that Col scaffold was not preferable for hiPSC culture, while the neurally committed population after seeding on Col scaffolds revealed good adhesion, viability, proliferation, along with sustaining markers of neuronal and glial differentiation. The Col scaffold-based 3D culture of hiPSC-NSCs may serve as a research tool for further translational studies.

Application of CRISPR/Cas9 technologies combined with iPSCs in the study and treatment of retinal degenerative diseases.

Retinal degeneration diseases, such as age-related macular degeneration and retinitis pigmentosa, affect millions of people worldwide and are major causes of irreversible blindness. Effective treatments for retinal degeneration, including drug therapy, gene augmentation or transplantation approaches, have been widely investigated. Nevertheless, more research should be dedicated to therapeutic methods to improve future clinical treatments. Recently, with the rapid development of genome-editing technology, gene therapy has become a potentially effective treatment for retinal degeneration diseases. A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been developed as a powerful genome-editing tool in ophthalmic studies. The CRISPR/Cas9 system has been widely applied in basic research to develop animal models and gene therapies in vivo. With the ability to self-renew and the potential to differentiate into different types of cells, induced pluripotent stem cells (iPSCs) have already been used as a promising tool for understanding disease pathophysiology and evaluating the effect of drug and gene therapeutics. iPSCs are also a cell source for autologous transplantation. In this review, we compared genome-editing strategies and highlighted the advantages and concerns of the CRISPR/Cas9 system. Moreover, the latest progress and applications of the CRISPR/Cas9 system and its combination with iPSCs for the treatment of retinal degenerative diseases are summarized.

Zinc Finger Nucleases Targeting The β-Globin Locus Drive Efficient Correction Of The Sickle Mutation In CD34+ Cells

Despite major improvements in clinical care and advances in understanding of its complex pathophysiology, sickle cell disease (SCD) continues to be a significant cause of morbidity and early mortality. Allogeneic hematopoietic stem cell transplant (HSCT) can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, allogeneic HSCT is limited by the availability of well-matched donors and the immunological complications of graft rejection and graft-versus-host disease that can occur, especially for the more than 80% of patients who lack an HLA-identical sibling donor. Gene therapy could provide a way to cure SCD; however, the current approaches use integrating lentiviral vectors, and therefore carry a risk of insertional oncogenesis. An alternative approach is to use site-specific nucleases to correct the patients' own cells, obviating the need for allogeneic HSCT and the use of randomly integrating vectors.Zinc finger nucleases (ZFNs) offer a possible way to achieve successful gene therapy by site-specifically and permanently modifying the endogenous gene in hematopoietic stem cells (HSCs). These engineered nucleases create a site-specific, double strand break upon dimerization. If a homologous donor molecule is co-introduced which contains the normal β-globin sequence at the site of the sickle mutation, the cells may undergo homology-directed repair to correct the mutation and restore functional hemoglobin production. With this aim in mind, we have designed and tested ZFN pairs targeting the β-globin locus along with a donor template that restores the normal β-globin gene sequence while simultaneously introducing a silent base pair change that generates a restriction enzyme site for analysis. These components have led to high levels of site-specific base-pair modification in introducing the sickle mutation at the normal β-globin locus in K562 cells (upwards of 45%). Using electroporation, we delivered the ZFNs as mRNA to cord blood-derived (CB) CD34+ cells which resulted in up to 30% allelic disruption as measured by the Surveyor Nuclease assay. To achieve gene correction, the ZFNs were again delivered as mRNA and the donor template was delivered as an integrase defective lentiviral vector (IDLV). Based on pyrosequencing data, this delivery method resulted in up to 10% gene correction (the correct nucleotide replacing the sickle mutation in β-globin). Importantly, in the clinically relevant cell source, namely CD34+ cells isolated from SCD patient bone marrow, these gene modification frequencies were maintained, resulting in up to 7% correction using this multi-modal delivery strategy. These data set the stage for further investigations, including ongoing studies in a humanized mouse model. Efficient correction of the sickle mutation in HSC may provide an excellent stem cell source for autologous transplantation for SCD. Disclosures:Cost:Sangamo BioSciences: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.

Dopaminergic Neuronal Conversion from Adult Rat Skeletal Muscle-Derived Stem Cells In Vitro

Muscle-derived stem cells reside in the skeletal muscle tissues and are known for their multipotency to differentiate toward the mesodermal lineage. Recent studies have demonstrated their capacity of neuroectodermal differentiation, including neurons and astrocytes. In this study, we investigated the possibility of dopaminergic neuronal conversion from adult rat skeletal muscle-derived stem cells. Using a neurosphere protocol, muscle-derived stem cells form neurosphere-like cell clusters after cultivation as a suspension, displaying an obvious expression of nestin and a remarkable down-regulation of myogenic associated factors desmin, MyoD, Myf5 and myogenin. Subsequently, these neurosphere-like cell clusters were further directed to dopaminergic differentiation through two major induction steps, patterning to midbrain progenitors with sonic hedgehog and fibroblast growth factor 8, followed by the differentiation to dopaminergic neurons with neurotrophic factors (glial cell line-derived neurotrophic factor) and chemicals (ascorbic acid, forskolin). After the differentiation, these cells expressed tyrosine hydroxylase, dopamine transporter, dopamine D1 receptor and synapse-associated protein synapsin I. Several genes, Nurr1, Lmx1b, and En1, which are critically related with the development of dopaminergic neurons, were also significantly up-regulated. The present results indicate that adult skeletal muscle-derived stem cells could provide a promising cell source for autologous transplantation for neurodegenerative diseases in the future, especially the Parkinson's disease.

Higher incidence of relapse in patients with acute myelocytic leukemia infused with higher doses of CD34+ cells from leukapheresis products autografted during the first remission

AbstractThe stem cell source for autologous transplantation has shifted from bone marrow to peripheral blood (PB). We previously showed that relapse incidence in patients with acute myelocytic leukemia autografted in first remission (CR1) was greater with PB than bone marrow, and a poorer outcome was associated with a shorter CR1 to PB transplantation interval (≤ 80 days). Leukemic and normal progenitors are CD34+ and can be concomitantly mobilized; we assessed whether an association exists between the infused CD34+ cell dose and outcome. The infused CD34+ cell doses were available for 772 patients autografted more than 80 days after CR1 and were categorized by percentiles. We selected the highest quintile (> 7.16 × 106/kg) as the cutoff point. By multivariate analysis, relapse was more probable in patients who received the highest dose (hazard ratio = 1.48; 95% confidence interval, 1.12-1.95; P = .005), and leukemia-free survival was worse (hazard ratio = 0.72; 95% confidence interval, 0.55-0.93; P = .01). In conclusion, in patients autografted in first remission, relapse was higher and leukemia-free survival lower for those who received the highest CD34+ PB doses.

Peripheral Blood or Bone Marrow As Hematopoietic Stem Cell Source for Autologous Transplantation in Acute Myeloid Leukemia?

Peripheral Blood or Bone Marrow As Hematopoietic Stem Cell Source for Autologous Transplantation in Acute Myeloid Leukemia?

Introducing Transcription Factors to Multipotent Mesenchymal Stem Cells: Making Transdifferentiation Possible

Multipotent mesenchymal stem cells (MSCs) represent a promising autologous source for regenerative medicine. Because MSCs can be isolated from adult tissues, they represent an attractive cell source for autologous transplantation. A straightforward therapeutic strategy in the field of stem cell-based regenerative medicine is the transplantation of functional differentiated cells as cell replacement for the lost or defective cells affected by disease. However, this strategy requires the capacity to regulate stem cell differentiation toward the desired cell fate. This therapeutic approach assumes the capability to direct MSC differentiation toward diverse cell fates, including those outside the mesenchymal lineage, a process termed transdifferentiation. The capacity of MSCs to undergo functional transdifferentiation has been questioned over the years. Nonetheless, recent studies support that genetic manipulation can serve to promote transdifferentiation. Specifically, forced expression of certain transcription factors can lead to reprogramming and alter cell fate. Using such a method, fully differentiated lymphocytes have been reprogrammed to become macrophages and, remarkably, somatic cells have been reprogrammed to become embryonic stem-like cells. In this review, we discuss the past and current research aimed at transdifferentiating MSCs, a process with applications that could revolutionize regenerative medicine.

Lentiviral Delivery of LMX1a Enhances Dopaminergic Phenotype in Differentiated Human Bone Marrow Mesenchymal Stem Cells

Human mesenchymal stem cells (MSCs) reside in the bone marrow and are known for their ability to differentiate along the mesenchymal lineage (fat, bone, and cartilage). Recent works have suggested the possibility that these cells are also capable of differentiating toward the neuroectodermal lineage. Using lentiviral gene delivery, we sought to reprogram the bone marrow-derived MSCs toward dopaminergic differentiation through delivery of LMX1a, which was reported to be a key player in dopaminergic differentiation in both developmental animal models and embryonic stem cells. Transduction of cells with fluorescent reporter genes confirmed efficiency of gene delivery. On incubation of the LMX1a transduced cells in differentiation medium, the LMX1a protein was concentrated in the cells' nuclei and specific dopaminergic developmental genes were upregulated. Moreover, the transduced cells expressed higher levels of tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis, and secreted significantly higher level of dopamine in comparison to nontransduced cells. We hereby present a novel strategy to facilitate the dopaminergic differentiation of bone marrow-derived MSCs as a possible cell source for autologous transplantation for Parkinsonian patients in the future.

Subcutaneous transplantation of autologous oral mucosal epithelial cell sheets fabricated on temperature‐responsive culture dishes

The oral mucosa is an attractive cell source for autologous transplantation in human patients who require regenerative therapies of various epithelia. However, the time-course of cellular changes in transplanted oral mucosal epithelia at ectopic sites remains poorly understood. By applying a rat model, we analyzed phenotypic changes in oral mucosal epithelial cell sheets after harvest from temperature-responsive culture dishes and subsequent autologous subcutaneous transplantation. We used monoclonal antibodies to identify epithelial-specific cytokeratins 4, 10, 13, and 14, the stem/progenitor cell marker p63, and proliferating cell nuclear antigen, within the regenerated tissues. Transplanted oral mucosal epithelial cell sheets proliferated during the first week after grafting in conjunction with host inflammation, but then began to degenerate afterward with complete disappearance after 3 weeks. Our findings suggest that host subcutaneous tissues support proliferation and differentiation of the oral mucosal epithelial cell sheets, but are unable to promote maintenance of stem and progenitor cells and therefore cannot produce long-term survivability.

Comparison of the Fenwal Amicus and Fresenius Com.Tec cell separators for autologous peripheral blood progenitor cell collection

Peripheral blood progenitor cells (PBPC) are commonly used as a stem cell source for autologous transplantation. This study was undertaken to evaluate blood cell separators with respect to separation results and content of the harvest. Forty autologous PBPC collections in patients with hematological malignancies were performed with either the Amicus or the COM.TEC cell separators. The median product volume was lower with the Amicus compared to the COM.TEC (125 mL vs. 300 mL; p < 0.001). There was no statistically significant difference in the median number of CD34+ cell/kg in product between the Amicus and the COM.TEC (3.0 × 10 6 vs. 4.1 × 10 6; p = 0.129). There was a statistically higher mean volume of ACD used in collections on the Amicus compared to the COM.TEC (1040 ± 241 mL vs. 868 ± 176 mL; p = 0.019). There was a statistical difference in platelet (PLT) contamination of the products between the Amicus and the COM.TEC (0.3 × 10 11 vs. 1.1 × 10 11; p < 0.001). The median % decrease in PB PLT count was statistically higher in the COM.TEC compared to the Amicus instruments (18.5% vs. 9.5%; p = 0.028). In conclusion, both instruments collected PBPCs efficiently. However, Amicus has the advantage of lower PLT contamination in the product, and less decrease in PB platelet count with lower product volume in autologous setting.

Beating is necessary for transdifferentiation of skeletal muscle-derived cells into cardiomyocytes.

Cell transplantation could be a potential therapy for heart damage. Skeletal myoblasts have been expected to be a good cell source for autologous transplantation; however, the safety and efficacy of their transplantation are still controversial. Recent studies have revealed that skeletal muscle possesses the stem cell population that is distinct from myoblasts. To elucidate whether skeletal muscle stem cells can transdifferentiate into cardiomyocytes, we cocultured skeletal muscle cells isolated from transgenic mice expressing green fluorescent protein with cardiomyocytes of neonatal rats. Skeletal muscle-derived cells expressed cardiac-specific proteins such as cardiac troponin T and atrial natriuretic peptide as well as cardiac-enriched transcription factors such as Nkx2E (formerly called Csx/Nkx2.5) and GATA4 by coculture with cardiomyocytes. Skeletal muscle-derived cells also expressed cadherin and connexin 43 at the junctions with neighboring cardiomyocytes. Cardiomyocyte-like action potentials were recorded from beating skeletal muscle-derived cells. Treatment of nifedipine or culture in Ca2+-free media suppressed contraction of cardiomyocytes and inhibited skeletal muscle cells to express cardiac-specific proteins. Cyclic stretch completely restored this inhibitory effect. These results suggest that some part of skeletal muscle cells can transdifferentiate into cardiomyocytes and that direct cell-to-cell contact and contraction of neighboring cardiomyocytes are important for the transdifferentiation.