Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4–contaminated human testicular cell suspensions was performed to isolate EpCAM+/HLA-ABC–/CD49e– (putative spermatogonia) and EpCAM–/HLA-ABC+/CD49e+ (putative MOLT-4) cell fractions. The EpCAM+/HLA-ABC–/CD49e– fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to–nude mouse xenotransplantation. The EpCAM–/HLA-ABC+/CD49e+ fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression.
Serena L. Dovey, Hanna Valli, Brian P. Hermann, Meena Sukhwani, Julia Donohue, Carlos A. Castro, Tianjiao Chu, Joseph S. Sanfilippo, Kyle E. Orwig
Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurr1+ stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.
Yosif M. Ganat, Elizabeth L. Calder, Sonja Kriks, Jenny Nelander, Edmund Y. Tu, Fan Jia, Daniela Battista, Neil Harrison, Malin Parmar, Mark J. Tomishima, Urs Rutishauser, Lorenz Studer
Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. Neural derivatives of human embryonic stem cells (hESCs) are currently being used for cell therapy in 3 clinical trials. However, hESCs are prone to genomic instability, which could limit their clinical utility. Here, we report that neural differentiation of hESCs systematically produced a neural stem cell population that could be propagated for more than 50 passages without entering senescence; this was true for all 6 hESC lines tested. The apparent spontaneous loss of evolution toward normal senescence of somatic cells was associated with a jumping translocation of chromosome 1q. This chromosomal defect has previously been associated with hematologic malignancies and pediatric brain tumors with poor clinical outcome. Neural stem cells carrying the 1q defect implanted into the brains of rats failed to integrate and expand, whereas normal cells engrafted. Our results call for additional quality controls to be implemented to ensure genomic integrity not only of undifferentiated pluripotent stem cells, but also of hESC derivatives that form cell therapy end products, particularly neural lines.
Christine Varela, Jérôme Alexandre Denis, Jérôme Polentes, Maxime Feyeux, Sophie Aubert, Benoite Champon, Geneviève Piétu, Marc Peschanski, Nathalie Lefort
Spinal cord injury (SCI) often leads to persistent functional deficits due to loss of neurons and glia and to limited axonal regeneration after injury. Here we report that transplantation of human dental pulp stem cells into the completely transected adult rat spinal cord resulted in marked recovery of hind limb locomotor functions. Transplantation of human bone marrow stromal cells or skin-derived fibroblasts led to substantially less recovery of locomotor function. The human dental pulp stem cells exhibited three major neuroregenerative activities. First, they inhibited the SCI-induced apoptosis of neurons, astrocytes, and oligodendrocytes, which improved the preservation of neuronal filaments and myelin sheaths. Second, they promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, including chondroitin sulfate proteoglycan and myelin-associated glycoprotein, via paracrine mechanisms. Last, they replaced lost cells by differentiating into mature oligodendrocytes under the extreme conditions of SCI. Our data demonstrate that tooth-derived stem cells may provide therapeutic benefits for treating SCI through both cell-autonomous and paracrine neuroregenerative activities.
Kiyoshi Sakai, Akihito Yamamoto, Kohki Matsubara, Shoko Nakamura, Mami Naruse, Mari Yamagata, Kazuma Sakamoto, Ryoji Tauchi, Norimitsu Wakao, Shiro Imagama, Hideharu Hibi, Kenji Kadomatsu, Naoki Ishiguro, Minoru Ueda
Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell–based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.
Yong-Hee Rhee, Ji-Yun Ko, Mi-Yoon Chang, Sang-Hoon Yi, Dohoon Kim, Chun-Hyung Kim, Jae-Won Shim, A-Young Jo, Byung-Woo Kim, Hyunsu Lee, Suk-Ho Lee, Wonhee Suh, Chang-Hwan Park, Hyun-Chul Koh, Yong-Sung Lee, Robert Lanza, Kwang-Soo Kim, Sang-Hun Lee
The directed differentiation of iPS and ES cells into definitive endoderm (DE) would allow the derivation of otherwise inaccessible progenitors for endodermal tissues. However, a global comparison of the relative equivalency of DE derived from iPS and ES populations has not been performed. Recent reports of molecular differences between iPS and ES cells have raised uncertainty as to whether iPS cells could generate autologous endodermal lineages in vitro. Here, we show that both mouse iPS and parental ES cells exhibited highly similar in vitro capacity to undergo directed differentiation into DE progenitors. With few exceptions, both cell types displayed similar surges in gene expression of specific master transcriptional regulators and global transcriptomes that define the developmental milestones of DE differentiation. Microarray analysis showed considerable overlap between the genetic programs of DE derived from ES/iPS cells in vitro and authentic DE from mouse embryos in vivo. Intriguingly, iPS cells exhibited aberrant silencing of imprinted genes known to participate in endoderm differentiation, yet retained a robust ability to differentiate into DE. Our results show that, despite some molecular differences, iPS cells can be efficiently differentiated into DE precursors, reinforcing their potential for development of cell-based therapies for diseased endoderm-derived tissues.
Constantina Christodoulou, Tyler A. Longmire, Steven S. Shen, Alice Bourdon, Cesar A. Sommer, Paul Gadue, Avrum Spira, Valerie Gouon-Evans, George J. Murphy, Gustavo Mostoslavsky, Darrell N. Kotton
Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising candidate cell sources for regenerative medicine. However, despite the common ability of hiPSCs and hESCs to differentiate into all 3 germ layers, their functional equivalence at the single cell level remains to be demonstrated. Moreover, single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here, we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs, suggesting that hiPSCs occupy an alternate, less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exercised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs, particularly when producing differentiated cells for regenerative medicine aims.
Kazim H. Narsinh, Ning Sun, Veronica Sanchez-Freire, Andrew S. Lee, Patricia Almeida, Shijun Hu, Taha Jan, Kitchener D. Wilson, Denise Leong, Jarrett Rosenberg, Mylene Yao, Robert C. Robbins, Joseph C. Wu
Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells.
Kunihiko Hiramatsu, Satoru Sasagawa, Hidetatsu Outani, Kanako Nakagawa, Hideki Yoshikawa, Noriyuki Tsumaki
To be of therapeutic use, autologous stem cells derived from patients with inherited genetic disorders require genetic modification via gene repair or insertion. Here, we present proof of principle that, for diseases associated with dominant alleles (gain-of-function or haploinsufficient loss-of-function), disease allele–free ES cells can be derived from afflicted individuals without genome manipulation. This approach capitalizes on the derivation of uniparental cells, such as parthenogenetic (PG) ES cell lines from disease allele–free gametes. Diploid mammalian uniparental embryos with only maternally (oocyte-) or paternally (sperm-)derived genomes fail early in development due to the nonequivalence of parental genomes caused by genomic imprinting. However, these uniparental embryos develop to the blastocyst stage, allowing the derivation of ES cell lines. Using a mouse model for dominant beta-thalassemia, we developed disease allele–free PG ES cell lines from the oocytes of affected animals. Phenotype correction was obtained in donor-genotype recipients after transplantation of in vitro hematopoietic ES cell derivatives. This genetic correction strategy without gene targeting is potentially applicable to any dominant disease. It could also be the sole approach for larger or more complex mutations that cannot be corrected by homologous recombination.
Sigrid Eckardt, N. Adrian Leu, Ashley Yanchik, Seigo Hatada, Michael Kyba, K. John McLaughlin
Human induced pluripotent stem (iPS) cells hold great promise for advancements in developmental biology, cell-based therapy, and modeling of human disease. Here, we examined the use of human iPS cells for modeling inherited metabolic disorders of the liver. Dermal fibroblasts from patients with various inherited metabolic diseases of the liver were used to generate a library of patient-specific human iPS cell lines. Each line was differentiated into hepatocytes using what we believe to be a novel 3-step differentiation protocol in chemically defined conditions. The resulting cells exhibited properties of mature hepatocytes, such as albumin secretion and cytochrome P450 metabolism. Moreover, cells generated from patients with 3 of the inherited metabolic conditions studied in further detail (α1-antitrypsin deficiency, familial hypercholesterolemia, and glycogen storage disease type 1a) were found to recapitulate key pathological features of the diseases affecting the patients from which they were derived, such as aggregation of misfolded α1-antitrypsin in the endoplasmic reticulum, deficient LDL receptor–mediated cholesterol uptake, and elevated lipid and glycogen accumulation. Therefore, we report a simple and effective platform for hepatocyte generation from patient-specific human iPS cells. These patient-derived hepatocytes demonstrate that it is possible to model diseases whose phenotypes are caused by pathological dysregulation of key processes within adult cells.
S. Tamir Rashid, Sebastien Corbineau, Nick Hannan, Stefan J. Marciniak, Elena Miranda, Graeme Alexander, Isabel Huang-Doran, Julian Griffin, Lars Ahrlund-Richter, Jeremy Skepper, Robert Semple, Anne Weber, David A. Lomas, Ludovic Vallier
The ability to generate induced pluripotent stem (iPS) cells from a patient’s somatic cells has provided a foundation for organ regeneration without the need for immune suppression. However, it has not been established that the differentiated progeny of iPS cells can effectively reverse failure of a vital organ. Here, we examined whether iPS cell–derived hepatocytes have both the functional and proliferative capabilities needed for liver regeneration in mice with fumarylacetoacetate hydrolase deficiency. To avoid biases resulting from random genomic integration, we used iPS cells generated without viruses. To exclude compensation by hepatocytes not derived from iPS cells, we generated chimeric mice in which all hepatocytes were iPS cell derived. In vivo analyses showed that iPS cells were intrinsically able to differentiate into fully mature hepatocytes that provided full liver function. The iPS cell–derived hepatocytes also replicated the unique proliferative capabilities of normal hepatocytes and were able to regenerate the liver after transplantation and two-thirds partial hepatectomy. Thus, our results establish the feasibility of using iPS cells generated in a clinically acceptable fashion for rapid and stable liver regeneration.
Silvia Espejel, Garrett R. Roll, K. John McLaughlin, Andrew Y. Lee, Jenny Y. Zhang, Diana J. Laird, Keisuke Okita, Shinya Yamanaka, Holger Willenbring
The body’s capacity to restore damaged neural networks in the injured CNS is severely limited. Although various treatment regimens can partially alleviate spinal cord injury (SCI), the mechanisms responsible for symptomatic improvement remain elusive. Here, using a mouse model of SCI, we have shown that transplantation of neural stem cells (NSCs) together with administration of valproic acid (VPA), a known antiepileptic and histone deacetylase inhibitor, dramatically enhanced the restoration of hind limb function. VPA treatment promoted the differentiation of transplanted NSCs into neurons rather than glial cells. Transsynaptic anterograde corticospinal tract tracing revealed that transplant-derived neurons reconstructed broken neuronal circuits, and electron microscopic analysis revealed that the transplant-derived neurons both received and sent synaptic connections to endogenous neurons. Ablation of the transplanted cells abolished the recovery of hind limb motor function, confirming that NSC transplantation directly contributed to restored motor function. These findings raise the possibility that epigenetic status in transplanted NSCs can be manipulated to provide effective treatment for SCI.
Masahiko Abematsu, Keita Tsujimura, Mariko Yamano, Michiko Saito, Kenji Kohno, Jun Kohyama, Masakazu Namihira, Setsuro Komiya, Kinichi Nakashima
The mechanisms of BM hematopoietic stem/progenitor cell (HSPC) adhesion, engraftment, and mobilization remain incompletely identified. Here, using WT and transgenic mice, we have shown that membrane-anchored plasminogen activator, urokinase receptor (MuPAR) marks a subset of HSPCs and promotes the preservation of the size of this pool of cells in the BM. Loss or inhibition of MuPAR increased HSPC proliferation and impaired their homing, engraftment, and adhesion to the BM microenvironment. During mobilization, MuPAR was inactivated by plasmin via proteolytic cleavage. Cell-autonomous loss of the gene encoding MuPAR also impaired long-term engraftment and multilineage repopulation in primary and secondary recipient mice. These findings identify MuPAR and plasmin as regulators of the proliferation, marrow pool size, homing, engraftment, and mobilization of HSPCs and possibly also of HSCs.
Marc Tjwa, Nicolai Sidenius, Rute Moura, Sandra Jansen, Koen Theunissen, Annapaola Andolfo, Maria De Mol, Mieke Dewerchin, Lieve Moons, Francesco Blasi, Catherine Verfaillie, Peter Carmeliet
The esophageal epithelium is a prototypical stratified squamous epithelium that exhibits an exquisite equilibrium between proliferation and differentiation. After basal cells proliferate, they migrate outward toward the luminal surface, undergo differentiation, and eventually slough due to apoptosis. The identification and characterization of stem cells responsible for the maintenance of the esophageal epithelium remains elusive. Here, we employed Hoechst dye extrusion and BrdU label–retaining assays to identify in mice a potential esophageal stem cell population that localizes to the basal cell compartment. The self-renewing capacity of this population was characterized using a clonogenic assay and a 3D organotypic culture model. The putative esophageal stem cells were also capable of epithelial reconstitution in vivo in direct esophageal epithelial injury models. In both the 3D organotypic culture and direct mucosal injury models, the putative stem cells gave rise to undifferentiated and differentiated cells. These studies therefore provide a basis for understanding the regenerative capacity and biology of the esophageal epithelium when it is faced with injurious insults.
Jiri Kalabis, Kenji Oyama, Takaomi Okawa, Hiroshi Nakagawa, Carmen Z. Michaylira, Douglas B. Stairs, Jose-Luiz Figueiredo, Umar Mahmood, J. Alan Diehl, Meenhard Herlyn, Anil K. Rustgi
Dopamine (DA) cell replacement therapy in Parkinson disease (PD) can be achieved using human fetal mesencephalic tissue; however, limited tissue availability has hindered further developments. Embryonic stem cells provide a promising alternative, but poor survival and risk of teratoma formation have prevented their clinical application. We present here a method for generating large numbers of DA neurons based on expanding and differentiating ventral midbrain (VM) neural stem cells/progenitors in the presence of key signals necessary for VM DA neuron development. Mouse VM neurospheres (VMNs) expanded with FGF2, differentiated with sonic hedgehog and FGF8, and transfected with Wnt5a (VMN-Wnt5a) generated 10-fold more DA neurons than did conventional FGF2-treated VMNs. VMN-Wnt5a cells exhibited the transcriptional and biochemical profiles and intrinsic electrophysiological properties of midbrain DA cells. Transplantation of these cells into parkinsonian mice resulted in significant cellular and functional recovery. Importantly, no tumors were detected and only a few transplanted grafts contained sporadic nestin-expressing progenitors. Our findings show that Wnt5a improves the differentiation and functional integration of stem cell–derived DA neurons in vivo and define Wnt5a-treated neural stem cells as an efficient and safe source of DA neurons for cell replacement therapy in PD.
Clare L. Parish, Gonçalo Castelo-Branco, Nina Rawal, Jan Tonnesen, Andreas Toft Sorensen, Carmen Salto, Merab Kokaia, Olle Lindvall, Ernest Arenas
Jeffrey J. Ross, Zhigang Hong, Ben Willenbring, Lepeng Zeng, Brett Isenberg, Eu Han Lee, Morayma Reyes, Susan A. Keirstead, E. Kenneth Weir, Robert T. Tranquillo, Catherine M. Verfaillie
Endothelial progenitor cells (EPCs) are essential in vasculogenesis and wound healing, but their circulating and wound level numbers are decreased in diabetes. This study aimed to determine mechanisms responsible for the diabetic defect in circulating and wound EPCs. Since mobilization of BM EPCs occurs via eNOS activation, we hypothesized that eNOS activation is impaired in diabetes, which results in reduced EPC mobilization. Since hyperoxia activates NOS in other tissues, we investigated whether hyperoxia restores EPC mobilization in diabetic mice through BM NOS activation. Additionally, we studied the hypothesis that impaired EPC homing in diabetes is due to decreased wound level stromal cell–derived factor–1α (SDF-1α), a chemokine that mediates EPC recruitment in ischemia. Diabetic mice showed impaired phosphorylation of BM eNOS, decreased circulating EPCs, and diminished SDF-1α expression in cutaneous wounds. Hyperoxia increased BM NO and circulating EPCs, effects inhibited by the NOS inhibitor N-nitro-l-arginine-methyl ester. Administration of SDF-1α into wounds reversed the EPC homing impairment and, with hyperoxia, synergistically enhanced EPC mobilization, homing, and wound healing. Thus, hyperoxia reversed the diabetic defect in EPC mobilization, and SDF-1α reversed the diabetic defect in EPC homing. The targets identified, which we believe to be novel, can significantly advance the field of diabetic wound healing.
Katherine A. Gallagher, Zhao-Jun Liu, Min Xiao, Haiying Chen, Lee J. Goldstein, Donald G. Buerk, April Nedeau, Stephen R. Thom, Omaida C. Velazquez
Smooth muscle formation and function are critical in development and postnatal life. Hence, studies aimed at better understanding SMC differentiation are of great importance. Here, we report that multipotent adult progenitor cells (MAPCs) isolated from rat, murine, porcine, and human bone marrow demonstrate the potential to differentiate into cells with an SMC-like phenotype and function. TGF-β1 alone or combined with PDGF-BB in serum-free medium induces a temporally correct expression of transcripts and proteins consistent with smooth muscle development. Furthermore, SMCs derived from MAPCs (MAPC-SMCs) demonstrated functional L-type calcium channels. MAPC-SMCs entrapped in fibrin vascular molds became circumferentially aligned and generated force in response to KCl, the L-type channel opener FPL64176, or the SMC agonists 5-HT and ET-1, and exhibited complete relaxation in response to the Rho-kinase inhibitor Y-27632. Cyclic distention (5% circumferential strain) for 3 weeks increased responses by 2- to 3-fold, consistent with what occurred in neonatal SMCs. These results provide evidence that MAPC-SMCs are phenotypically and functionally similar to neonatal SMCs and that the in vitro MAPC-SMC differentiation system may be an ideal model for the study of SMC development. Moreover, MAPC-SMCs may lend themselves to tissue engineering applications.
Jeffrey J. Ross, Zhigang Hong, Ben Willenbring, Lepeng Zeng, Brett Isenberg, Eu Han Lee, Morayma Reyes, Susan A. Keirstead, E. Kenneth Weir, Robert T. Tranquillo, Catherine M. Verfaillie
Vision loss associated with ischemic diseases such as retinopathy of prematurity and diabetic retinopathy are often due to retinal neovascularization. While significant progress has been made in the development of compounds useful for the treatment of abnormal vascular permeability and proliferation, such therapies do not address the underlying hypoxia that stimulates the observed vascular growth. Using a model of oxygen-induced retinopathy, we demonstrate that a population of adult BM–derived myeloid progenitor cells migrated to avascular regions of the retina, differentiated into microglia, and facilitated normalization of the vasculature. Myeloid-specific hypoxia-inducible factor 1α (HIF-1α) expression was required for this function, and we also demonstrate that endogenous microglia participated in retinal vascularization. These findings suggest what we believe to be a novel therapeutic approach for the treatment of ischemic retinopathies that promotes vascular repair rather than destruction.
Matthew R. Ritter, Eyal Banin, Stacey K. Moreno, Edith Aguilar, Michael I. Dorrell, Martin Friedlander
Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.
Andrea Hoffmann, Gadi Pelled, Gadi Turgeman, Peter Eberle, Yoram Zilberman, Hadassah Shinar, Keren Keinan-Adamsky, Andreas Winkel, Sandra Shahab, Gil Navon, Gerhard Gross, Dan Gazit