Li-Ying Sung1, 6, Shaorong Gao1, 6, Hongmei Shen2, Hui Yu2, Yifang Song2, Sadie L Smith1, Ching-Chien Chang1, Kimiko Inoue1, Lynn Kuo3, Jin Lian4, Ao Li5, X Cindy Tian1, David P Tuck5, Sherman M Weissman4, Xiangzhong Yang1 & Tao Cheng2

1-Center for Regenerative Biology and Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269, USA.
2-Cancer Stem Cell Program, University of Pittsburgh Cancer Institute and Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
3-Department of Statistics, University of Connecticut, Storrs, Connecticut 06269, USA.
4-Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
5-Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
6-These authors contributed equally to this work.

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer

Since the creation of Dolly via somatic cell nuclear transfer (SCNT)1, more than a dozen species of mammals have been cloned using this technology2. One hypothesis for the limited success of cloning via SCNT (1%–5%)3 is that the clones are likely to be derived from adult stem cells4. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors5, 6 and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells7, 8, 9, 10. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

Nature Genetics - 38, 1323 - 1328 (2006)

Yijen L. Wu, Qing Ye, Lesley M. Foley, T. Kevin Hitchens, Kazuya Sato, John B. Williams, and Chien Ho 

In situ labeling of immune cells with iron oxide particles: An approach to detect organ rejection by cellular MRI

In vivo cell tracking by MRI can provide means to observe biological processes and monitor cell therapy directly. Immune cells, e.g., macrophages, play crucial roles in many pathophysiological processes, including organ rejection, inflammation, autoimmune diseases, cancer, atherosclerotic plaque formation, numerous neurological disorders, etc. The current gold standard for diagnosing and staging rejection after organ transplantation is biopsy, which is not only invasive, but also prone to sampling errors. Here, we report a noninvasive approach using MRI to detect graft rejection after solid organ transplantation. In addition, we present the feasibility of imaging individual macrophages in vivo by MRI in a rodent heterotopic working-heart transplantation model using a more sensitive contrast agent, the micrometer-sized paramagnetic iron oxide particle, as a methodology to detect acute cardiac rejection.

Proceedings of the National Academy of Sciences 103: 1852-1857, 2006

Satdarshan P.S. Monga*, Mariah S. Hout, Matt J. Baun, Amanda Micsenyi*, Peggy Muller*, Lekha Tummalapalli*, Aarati R. Ranade, Jian-Hua Luo*, Stephen C. Strom* and Jörg C. Gerlach¶

From the Departments of Pathology,* Medicine (Gastroenterology), and Surgery and Bioengineering, McGowan Institute for Regenerative Medicine, and the Department of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania; and the Department of Surgery,¶ Charité-Campus Virchow, Humboldt University, Berlin, Germany

Mouse Fetal Liver Cells in Artificial Capillary Beds in Three-Dimensional Four-Compartment Bioreactors

Bioreactors containing porcine or adult human hepatocytes have been used to sustain acute liver failure patients until liver transplantation. However, prolonged function of adult hepatocytes has not been achieved due to compromised proliferation and viability of adult cells in vitro. We investigated the use of fetal hepatocytes as an alternative cell source in bioreactors. Mouse fetal liver cells from gestational day 17 possessed intermediate differentiation and function based on their molecular profile. When cultured in a three-dimensional four-compartment hollow fiber-based bioreactor for 3 to 5 weeks these cells formed neo-tissues that were characterized comprehensively. Albumin liberation, testosterone meta-bolism, and P450 induction were demonstrated. Histology showed predominant ribbon-like three-dimensional structures composed of hepatocytes between hollow fibers. High positivity for proliferating cell nuclear antigen and Ki-67 and low positivity for terminal dUTP nick-end labeling indicated robust cell proliferation and survival. Most cells within these ribbon arrangements were albumin-positive. In addition, cells in peripheral zones were simultaneously positive for -fetoprotein, cytokeratin-19, and c-kit, indicating their progenitor phenotype. Mesenchymal components including endothelial, stellate, and smooth muscle cells were also observed. Thus, fetal liver cells can survive, proliferate, differentiate, and function in a three-dimensional perfusion culture system while maintaining a progenitor pool, reflecting an important advance in hepatic tissue engineering. matrix (ECM), a tissue-engineered scaffold, recently demonstrated cardiomyocyte population after myocardial implantation. Surgical restoration of myocardium frequently uses Dacron as a myocardial patch. We hypothesized that an ECM-derived myocardial patch would provide a mechanical benefit not seen with Dacron.

American Journal of Pathology. 2005;167:1279-1292

Alexander Alexeev, Rolf Verberg and Anna C. Balazs

Modeling the interactions between deformable capsules rolling on a compliant surface

By integrating mesoscale models for hydrodynamics and micromechanics, we examine the fluid-driven motion of pairs of capsules on a compliant, adhesive substrate. The capsules, modeled as fluid filled elastic shells, represent ex vivo cells or polymeric microcapsules. We show that both the relative and the average velocities of two closely spaced, rolling capsules depends on the elasticity of the capsules, the adhesive interaction between the capsules and the substrate, and the compliance of the substrate. We first focused on a stiff surface and found that pairs of rigid capsules always separate from each other, while for deformable capsules, the dynamic behavior depends critically on the strength of the adhesive interaction. For strong adhesion to the substrate, the capsules again roll away from each other, while for a relatively weak adhesion, the capsules actually approach each other. In the case of soft substrates, any significant deformations of the surface that are caused by the capsules give rise to a force that propels the particles to move rapidly apart. Thus, in the case of strong adhesion between the capsules and the soft substrates, both rigid and flexible capsules are driven to separate. On the other hand, for weak adhesion, the elastic particles approach each other, similar to the behavior on stiff surfaces. These findings reveal that the interactions between the capsules are mediated by the nature of the underlying layer. We can harness this information to design surfaces that actively control the relative separation between the capsules. This could be utilized to regulate the motion of biological cells, as well as polymeric microcapsules, and thus, could prove to be useful in various biological assays or tissue engineering studies.

Soft Matter, 2006, 2, 499 - 509, DOI: 10.1039/b602417c

Paul V. Kochupura, MD; Evren U. Azeloglu, MS; Damon J. Kelly, MS; Sergey V. Doronin, PhD; Stephen F. Badylak, MD, PhD, DVM; Irvin B. Krukenkamp, MD; Ira S. Cohen, MD, PhD; Glenn R. Gaudette, PhD

Tissue-Engineered Myocardial Patch Derived From Extracellular Matrix Provides Regional Mechanical Function

Background—Extracellular matrix (ECM), a tissue-engineered scaffold, recently demonstrated cardiomyocyte population after myocardial implantation. Surgical restoration of myocardium frequently uses Dacron as a myocardial patch. We hypothesized that an ECM-derived myocardial patch would provide a mechanical benefit not seen with Dacron.

Methods and Results—Using a canine model, a full thickness defect in the right ventricle was repaired with either Dacron or ECM. A third group had no surgery and determined baseline RV function. Eight weeks later, global systolic function was assessed by the preload recruitable stroke work relationship. Regional systolic function was measured by systolic area contraction (SAC), calculated by high density mechanical mapping. Tau was used to assess global diastolic function. Recoil rate and diastolic shear were used as measures of regional diastolic function. After functional data acquisition, tissue was fixed for histological evaluation. Global systolic and diastolic functions were similar at baseline and after ECM and Dacron implantation. Regional systolic function was greater in the ECM group compared with the Dacron group.  Regional diastolic function was also greater in the ECM. Immunohistochemical analysis revealed cardiomyocytes in the ECM implant region, a finding not seen with Dacron.

Circulation. 2005;112 [suppl I]:I-144–I-149.

Todd Courtney, Michael S. Sacks, John Stankus, Jianjun Guan, William R. Wagner

Design and analysis of tissue engineering scaffolds that mimic soft tissue mechanical anisotropy

Tissue engineered constructs must exhibit tissue-like functional properties, including mechanical behavior comparable to the native tissues they are intended to replace. Moreover, the ability to reversibly undergo large strains can help to promote and guide tissue growth. Electrospun poly (ester urethane) ureas (ES-PEUU) are elastomeric and allow for the control of fiber diameter, porosity, and degradation rate. ES-PEUU scaffolds can be fabricated to have a well-aligned fiber network, which is important for applications involving mechanically anisotropic soft tissues.

We have developed ES-PEUU scaffolds under variable speed conditions and modeled the effects of fiber orientation on the macro-mechanical properties of the scaffold. To illustrate the ability to simulate native tissue mechanical behavior, we demonstrated that the high velocity spun scaffolds exhibited highly anisotropic mechanical properties closely resembling the native pulmonary heart valve leaflet. Moreover, use of the present fiber-level structural constitutive model allows for the determination of electrospinning conditions to tailor ES-PEUU scaffolds for specific soft tissue applications. The results of this study will help to provide the basis for rationally designed mechanically anisotropic soft tissue engineered implants.

Biomaterials, Volume 27, Issue 19, July 2006, Pages 3631-3638

Tavian, Manuela (a); Zheng, Bo (b); Oberlin, Estelle (a); Crisan, Mihaela (c); Sun, Bin (c); Huard, Johnny (b); Peault, Bruno (a,c)

a) Inserm U506, Hopital Paul Brousse, Villejuif, France

b) Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA

We have characterized the emerging hematopoietic system in the human embryo and fetus. Two embryonic organs, the yolk sac and aorta, support the primary emergence of hematopoietic stem cells (HSCs), but only the latter contributes lymphomyeloid stem cells for definitive, adult-type hematopoiesis.

A common feature of intra- and extraembryonic hematopoiesis is that in both locations hematopoietic cells emerge in close vicinity to vascular endothelial cells. We have provided evidence that a population of angiohematopoietic mesodermal stem cells, marked by the expression of flk-1 and the novel BB9/ACE antigen, migrate from the paraaortic splanchnopleura into the ventral part of the aorta, where they give rise to hemogenic endothelial cells and, in turn, hematopoietic cells. HSCs also appear to develop from endothelium in the embryonic liver and fetal bone marrow, albeit at a much lower frequency. This would imply that the organism does not function during its whole life on a stock of hematopoietic stem cells established in the early embryo, as is usually accepted. We next examined whether the vessel wall can contribute stem cells for other cell lineages, primarily in the model of adult skeletal muscle regeneration.

By immunohistochemistry and flow cytometry, we documented the existence in skeletal muscle, besides genuine endothelial and myogenic cells, of a subset of satellite cells that coexpress endothelial cell markers. This suggested the existence of a continuum of differentiation from vascular cells to endothelial cells that was confirmed in long-term culture. The regenerating capacity of these cells expressing both myogenic and endothelial markers is being investigated in skeletal and cardiac muscle, and the results are being compared with those generated by satellite cells.

Srikanth Ranganathan (1), Eric Williams (2), Philip Ganchev (2), Vanathi Gopalakrishnan (2), David Lacomis (3), Leo Urbinelli (4), Kristyn Newhall (4), Merit E. Cudkowicz (4), Robert H. Brown Jr. (5) and Robert Bowser (1)

1) Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
2) Center for Biomedical Informatics, University of Pittsburgh, Pittsburgh, PA, USA
3) Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
4) Neurology Clinical Trials Unit, Massachusetts General Hospital East, Charlestown, MA, USA
5) Day Neuromuscular Research Laboratory, Massachusetts General Hospital East, Charlestown, MA, USA

Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease, affecting one in every 40 000 individuals (Jackson and Bryan 1998). It typically affects individuals in their mid-50s and is characterized by rapidly progressive degeneration of motor neurons in the cerebral cortex, brainstem and spinal cord. The median survival in ALS is three to five years (Jackson and Bryan 1998; Cleveland and Rothstein 2001). ALS exists in both sporadic and familial forms. Familial ALS (FALS) comprises only 5–10% of all ALS cases.

Since amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons,  the study tested the hypothesis that proteomic analysis will identify protein biomarkers that provide insight into disease pathogenesis and are diagnostically useful.

To identify ALS specific biomarkers, the research team compared the proteomic profile of cerebrospinal fluid (CSF) from ALS and control subjects using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS).

The investigation identified 30 mass ion peaks with statistically significant (p < 0.01) differences between control and ALS subjects.  Initial analysis with a rule-learning algorithm yielded biomarker panels with diagnostic predictive value as subsequently assessed using an independent set of coded test subjects.

Three biomarkers were identified that are either decreased (transthyretin, cystatin C) or increased (carboxy-terminal fragment of neuroendocrine protein 7B2) in ALS CSF.

Hideki Oshima, Thomas R. Payne, Kenneth L. Urish, Tetsuro Sakai, Yiqun Ling, Burhan Gharaibeh, Kimimasa Tobita, Bradley B. Keller, James H. Cummins, and Johnny Huard

Myoblast transplantation for cardiac repair has generated beneficial results in both animals and humans; however, poor viability and poor engraftment of myoblasts after implantation in vivo limit their regeneration capacity. We and others have identified and isolated a subpopulation of skeletal muscle-derived stem cells (MDSCs) that regenerate skeletal muscle more effectively than myoblasts.

Here we report that in comparison with a myoblast population, MDSCs implanted into infracted hearts displayed greater and more persistent engraftment, induced more neoangiogenesis through graft expression of vascular endothelial growth factor, prevented cardiac remodeling, and elicited significant improvements in cardiac function. MDSCs also exhibited a greater ability to resist oxidative stress-induced apoptosis compared to myoblasts, which may partially explain the improved engraftment of MDSCs.

These findings indicate that MDSCs constitute an alternative to other myogenic cells for use in cardiac repair applications.

Amit N. Patel MD, MS, Luis Geffner MD, Roberto F. Vina MD, Jorge Saslavsky MD, Harold C. Urschel, Jr MD, Robert Kormos MD and Federico Benetti MD

This paper addresses autologous adult stem cell transplantation which has been touted as the latest tool in regenerative medical therapy. Its potential for use in cardiovascular disease has only recently been recognized. A randomized study was conducted by the authors using a novel epicardial technique to deploy stem cells as an adjuvant to conventional revascularization therapy in patients with congestive heart failure.

There were 20 patients enrolled in the study. Ten patients had successful subepicardial transplantation of autologous stem cells into ischemic myocardium. The other 10 patients, the control group, only had off-pump coronary artery bypass grafting. There were 8 male and 2 female subjects in each group. There were no perioperative arrhythmias or neurologic or ischemic myocardial events in either group.

2005

Chuanyue Wu

June 2006

Paul V. Kochupura, MD; Evren U. Azeloglu, MS; Damon J. Kelly, MS; Sergey V. Doronin, PhD; Stephen F. Badylak, MD, PhD, DVM; Irvin B. Krukenkamp, MD; Ira S. Cohen, MD, PhD; Glenn R. Gaudette, PhD

Tissue-Engineered Myocardial Patch Derived From Extracellular Matrix Provides Regional Mechanical Function

Background—Extracellular matrix (ECM), a tissue-engineered scaffold, recently demonstrated cardiomyocyte population after myocardial implantation. Surgical restoration of myocardium frequently uses Dacron as a myocardial patch. We hypothesized that an ECM-derived myocardial patch would provide a mechanical benefit not seen with Dacron.

Methods and Results—Using a canine model, a full thickness defect in the right ventricle was repaired with either Dacron or ECM. A third group had no surgery and determined baseline RV function. Eight weeks later, global systolic function was assessed by the preload recruitable stroke work relationship. Regional systolic function was measured by systolic area contraction (SAC), calculated by high density mechanical mapping. Tau was used to assess global diastolic function. Recoil rate and diastolic shear were used as measures of regional diastolic function. After functional data acquisition, tissue was fixed for histological evaluation. Global systolic and diastolic functions were similar at baseline and after ECM and Dacron implantation. Regional systolic function was greater in the ECM group compared with the Dacron group.  Regional diastolic function was also greater in the ECM. Immunohistochemical analysis revealed cardiomyocytes in the ECM implant region, a finding not seen with Dacron.

Circulation. 2005;112 [suppl I]:I-144–I-149.

2006

May

Todd Courtney, Michael S. Sacks, John Stankus, Jianjun Guan, William R. Wagner

Design and analysis of tissue engineering scaffolds that mimic soft tissue mechanical anisotropy

Tissue engineered constructs must exhibit tissue-like functional properties, including mechanical behavior comparable to the native tissues they are intended to replace. Moreover, the ability to reversibly undergo large strains can help to promote and guide tissue growth. Electrospun poly (ester urethane) ureas (ES-PEUU) are elastomeric and allow for the control of fiber diameter, porosity, and degradation rate. ES-PEUU scaffolds can be fabricated to have a well-aligned fiber network, which is important for applications involving mechanically anisotropic soft tissues.

We have developed ES-PEUU scaffolds under variable speed conditions and modeled the effects of fiber orientation on the macro-mechanical properties of the scaffold. To illustrate the ability to simulate native tissue mechanical behavior, we demonstrated that the high velocity spun scaffolds exhibited highly anisotropic mechanical properties closely resembling the native pulmonary heart valve leaflet. Moreover, use of the present fiber-level structural constitutive model allows for the determination of electrospinning conditions to tailor ES-PEUU scaffolds for specific soft tissue applications. The results of this study will help to provide the basis for rationally designed mechanically anisotropic soft tissue engineered implants.

Biomaterials, Volume 27, Issue 19, July 2006, Pages 3631-3638

April

Tavian, Manuela (a); Zheng, Bo (b); Oberlin, Estelle (a); Crisan, Mihaela (c); Sun, Bin (c); Huard, Johnny (b); Peault, Bruno (a,c)

a) Inserm U506, Hopital Paul Brousse, Villejuif, France

b) Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA

We have characterized the emerging hematopoietic system in the human embryo and fetus. Two embryonic organs, the yolk sac and aorta, support the primary emergence of hematopoietic stem cells (HSCs), but only the latter contributes lymphomyeloid stem cells for definitive, adult-type hematopoiesis.

A common feature of intra- and extraembryonic hematopoiesis is that in both locations hematopoietic cells emerge in close vicinity to vascular endothelial cells. We have provided evidence that a population of angiohematopoietic mesodermal stem cells, marked by the expression of flk-1 and the novel BB9/ACE antigen, migrate from the paraaortic splanchnopleura into the ventral part of the aorta, where they give rise to hemogenic endothelial cells and, in turn, hematopoietic cells. HSCs also appear to develop from endothelium in the embryonic liver and fetal bone marrow, albeit at a much lower frequency. This would imply that the organism does not function during its whole life on a stock of hematopoietic stem cells established in the early embryo, as is usually accepted. We next examined whether the vessel wall can contribute stem cells for other cell lineages, primarily in the model of adult skeletal muscle regeneration.

By immunohistochemistry and flow cytometry, we documented the existence in skeletal muscle, besides genuine endothelial and myogenic cells, of a subset of satellite cells that coexpress endothelial cell markers. This suggested the existence of a continuum of differentiation from vascular cells to endothelial cells that was confirmed in long-term culture. The regenerating capacity of these cells expressing both myogenic and endothelial markers is being investigated in skeletal and cardiac muscle, and the results are being compared with those generated by satellite cells.

March

Srikanth Ranganathan (1), Eric Williams (2), Philip Ganchev (2), Vanathi Gopalakrishnan (2), David Lacomis (3), Leo Urbinelli (4), Kristyn Newhall (4), Merit E. Cudkowicz (4), Robert H. Brown Jr. (5) and Robert Bowser (1)

1) Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
2) Center for Biomedical Informatics, University of Pittsburgh, Pittsburgh, PA, USA
3) Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
4) Neurology Clinical Trials Unit, Massachusetts General Hospital East, Charlestown, MA, USA
5) Day Neuromuscular Research Laboratory, Massachusetts General Hospital East, Charlestown, MA, USA

Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease, affecting one in every 40 000 individuals (Jackson and Bryan 1998). It typically affects individuals in their mid-50s and is characterized by rapidly progressive degeneration of motor neurons in the cerebral cortex, brainstem and spinal cord. The median survival in ALS is three to five years (Jackson and Bryan 1998; Cleveland and Rothstein 2001). ALS exists in both sporadic and familial forms. Familial ALS (FALS) comprises only 5–10% of all ALS cases.

Since amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons,  the study tested the hypothesis that proteomic analysis will identify protein biomarkers that provide insight into disease pathogenesis and are diagnostically useful.

To identify ALS specific biomarkers, the research team compared the proteomic profile of cerebrospinal fluid (CSF) from ALS and control subjects using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS).

The investigation identified 30 mass ion peaks with statistically significant (p < 0.01) differences between control and ALS subjects.  Initial analysis with a rule-learning algorithm yielded biomarker panels with diagnostic predictive value as subsequently assessed using an independent set of coded test subjects.

Three biomarkers were identified that are either decreased (transthyretin, cystatin C) or increased (carboxy-terminal fragment of neuroendocrine protein 7B2) in ALS CSF.

February

Hideki Oshima, Thomas R. Payne, Kenneth L. Urish, Tetsuro Sakai, Yiqun Ling, Burhan Gharaibeh, Kimimasa Tobita, Bradley B. Keller, James H. Cummins, and Johnny Huard

Myoblast transplantation for cardiac repair has generated beneficial results in both animals and humans; however, poor viability and poor engraftment of myoblasts after implantation in vivo limit their regeneration capacity. We and others have identified and isolated a subpopulation of skeletal muscle-derived stem cells (MDSCs) that regenerate skeletal muscle more effectively than myoblasts.

Here we report that in comparison with a myoblast population, MDSCs implanted into infracted hearts displayed greater and more persistent engraftment, induced more neoangiogenesis through graft expression of vascular endothelial growth factor, prevented cardiac remodeling, and elicited significant improvements in cardiac function. MDSCs also exhibited a greater ability to resist oxidative stress-induced apoptosis compared to myoblasts, which may partially explain the improved engraftment of MDSCs.

These findings indicate that MDSCs constitute an alternative to other myogenic cells for use in cardiac repair applications.

January

Amit N. Patel MD, MS, Luis Geffner MD, Roberto F. Vina MD, Jorge Saslavsky MD, Harold C. Urschel, Jr MD, Robert Kormos MD and Federico Benetti MD

This paper addresses autologous adult stem cell transplantation which has been touted as the latest tool in regenerative medical therapy. Its potential for use in cardiovascular disease has only recently been recognized. A randomized study was conducted by the authors using a novel epicardial technique to deploy stem cells as an adjuvant to conventional revascularization therapy in patients with congestive heart failure.

There were 20 patients enrolled in the study. Ten patients had successful subepicardial transplantation of autologous stem cells into ischemic myocardium. The other 10 patients, the control group, only had off-pump coronary artery bypass grafting. There were 8 male and 2 female subjects in each group. There were no perioperative arrhythmias or neurologic or ischemic myocardial events in either group.

2005

December

Chuanyue Wu