Regenerative Medicine: Progress in Stem Cell and Transplantation Research

Web Sites

Biomat.net
Biomat.net is aimed at linking the Biomaterials and Tissue Engineering community worldwide. This site provides updated news in the field, delivered through a monthly newsletter, and also a collection of selected internet links related to biomaterials science and tissue engineering, as well as relevant links to biomedical engineering, biology, medicine, and health sciences in general. Biomat.net facilities additionally include a glossary of related terms, a discussion forum, a job exchange section, and the directory of members, where research expertise can be searched.

California Institute for Regenerative Medicine
The California Institute for Regenerative Medicine was established in early 2005 following the passage of the California Stem Cell Research and Cures Initiative. The statewide ballot measure, which provided $3 billion in funding for stem cell research at California universities and research institutions, was approved by California voters on November 2, 2004, and called for the establishment of a new state agency to make grants and provide loans for stem cell research, research facilities and other vital research opportunities.

The mission of CIRM is to support and advance stem cell research and regenerative medicine under the highest ethical and medical standards for the discovery and development of cures, therapies, diagnostics and research technologies to relieve human suffering from chronic disease and injury.

Chinese Academy of Medical Science & Peking Union Medical College
The main task of the institute is searching for new drugs for treatment of commonly occurring diseases that seriously threaten people's health.

Chinese Academy of Sciences
The mission of the Chinese Academy of Sciences is to conduct research in basic and technological sciences; to undertake nationwide integrated surveys on natural resources and ecological environment; to provide the country with scientific data and advice for governmental decision-making, and to undertake government-assigned projects with regard to key S&T problems in the process of social and economic development; to initiate personnel training; and to promote China's high-tech enterprises by its active involvement in these areas.

European Society for Biomaterials
The European Society for Biomaterials (ESB) is non-profit scientific society whose main objective is to encourage progress in the field of biomaterials in all its aspects, including research, teaching, and clinical applications, as well as to foster any other related activity. It was founded in March 1976, and became a member of the International Union of Societies for Biomaterials Sciences and Engineering (IUS-BSE) at its conception in 1979. The ESB has approximately 750 members from 32 different countries worldwide.

Institute of Zoology, Chinese Academy of Sciences
The Institute of Zoology houses the State Key Laboratory of Biomembrane and Membrane Biotechnology.

National University of Singapore, Division of Bioengineering
The Division’s research focuses on the following core expertise: biomaterials, biomechanics, nano/micro-mechanics, biosignal processing, with application in areas of tissue engineering, biopharmaceutical engineering, biomedical imaging, biosensors and instrumentation, and medical devices.

Pittsburgh Tissue Engineering Initiative
The mission of PTEI is to improve the health of individuals by establishing the region as an internationally recognized center of excellence in research, education, and commercial development for the advancement of tissue-related medical therapies.

RegenerativeMedicine.Net
News of regenerative medicine research and therapies as reviewed by the experts at the McGowan Institute.

TERMIS
To accomplish its mission, the Tissue Engineering International & Regenerative Medicine Society (TERMIS) brings together the international community of persons engaged or interested in the field of tissue engineering and regenerative medicine, and promotes education and research within the field of tissue engineering and regenerative medicine through regular meetings, publications, and other forms of communication. The Society also serves as an international forum to promote the informed discussion of challenges and therapeutic benefits of the application of tissue engineering and regenerative medicine technologies.

Tissue Engineering Pages
Since 1995 the Tissue Engineering Pages Web site serves people interested in the fields of tissue engineering, regenerative medicine, and stem cell research. Meanwhile, this continually updated communication platform has become a well-known source of information to international experts in the field. The Tissue Engineering Pages provide up to date information on conferences, organizations, as wells as international tissue engineering projects. Editors and contributors are determined to continually improve this service for experts and people interested in this exciting field of biotechnology in the future.

Wake Forest University Institute for Regenerative Medicine
The goal of the institute is to apply the principles of regenerative medicine to repair or replaced diseased tissues and organs.

Journal Articles

Stefanie Dimmeler

Aicher A, Heeschen C, Sasaki K, et al. 2006. Low-energy shock wave for enhancing recruitment of endothelial progenitor cells: a new modality to increase efficacy of cell therapy in chronic hind limb ischemia. Circulation 114: 2823-2830. Full Text

Chavakis E, Urbich C, Dimmeler S. 2008. Homing and engraftment of progenitor cells: a prerequisite for cell therapy. J. Mol. Cell. Cardiol. 45: 514-522.

Dill T, Schächinger V, Rolf A, et al. 2009. Intracoronary administration of bone marrow-derived progenitor cells improves left ventricular function in patients at risk for adverse remodeling after acute ST-segment elevation myocardial infarction: results of the Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction study (REPAIR-AMI) cardiac magnetic resonance imaging substudy. Am. Heart J.157: 541-547.

Dimmeler S, Zeiher AM. 2009. Cell therapy of acute myocardial infarction: open questions. Cardiology 113: 155-160.

Sasaki K, Heeschen C, Aicher A, et al. 2006. Ex vivo pretreatment of bone marrow mononuclear cells with endothelial NO synthase enhancer AVE9488 enhances their functional activity for cell therapy. Proc. Natl. Acad. Sci. U.S.A.103: 14537-14541. Full Text

Anita Chong

Chidgey AP, Layton D, Trounson A, Boyd RL. 2008. Tolerance strategies for stem-cell-based therapies. Nature 453: 330-337.

Chong AS, Shen J, Tao J, et al. 2006. Reversal of diabetes in non-obese diabetic mice without spleen cell-derived beta cell regeneration. Science 311: 1774-1775.

Gregersen PK, Behrens TW. 2006. Genetics of autoimmune diseases--disorders of immune homeostasis. Nat. Rev. Genet.7: 917-928.

Keir ME, Liang SC, Guleria I, et al. 2006. Tissue expression of PD-L1 mediates peripheral T cell tolerance. J. Exp. Med.203: 883-895.

Kirk AD, Burkly LC, Batty DS, et al. 1999. Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nat. Med. 5: 686-693.

Morelli AE, Thomson AW. 2007. Tolerogenic dendritic cells and the quest for transplant tolerance. Nat. Rev. Immunol. 7: 610-621.

Niederkorn JY. 2006. See no evil, hear no evil, do no evil: the lessons of immune privilege. Nat. Immunol. 7: 354-359.

Sugamura K, Ishii N, Weinberg AD. 2004. Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40. Nat. Rev. Immunol.4: 420-431.

Yin D, Ma L, Zeng H, Shen J, Chong AS. 2002. Allograft tolerance induced by intact active bone co-transplantation and anti-CD40L monoclonal antibody therapy. Transplantation 74: 345-354.

Wei Han

Granville CA, Memmott RM, Balogh A et al. 2009. A central role for Foxp3+ regulatory T cells in K-Ras-driven lung tumorigenesis. PLoS One 4(3):e5061. Full Text

Zhang J, Xiang D, Zhu S, et al. 2008. Interleukin 1 receptor antagonist inhibits normal hematopoiesis and reduces lethality and bone marrow toxicity of 5-fluouracil in mouse. Biomed. Pharmacother. Oct 23. [Epub ahead of print]

Zhang Z, Du L, Xiang D, et al. 2009. Expression and purification of bioactive high-purity human midkine in Escherichia coli. J Zhejiang Univ Sci B 10: 79-86. Full Text

Tao Cheng

Boyer MJ, Cheng T. 2008. The CDK inhibitors: potential targets for therapeutic stem cell manipulations? Gene Ther. 15: 117-125.

Cheng T. 2008. Toward 'SMART' stem cells. Gene Ther. 15: 67-73.

Cheng T. 2004. Cell cycle inhibitors in normal and leukemic stem cells. Oncogene 23: 7256-7266.

Hu X, Shen H, Tian C, et al. 2009. Kinetics of normal hematopoietic stem and progenitor cells in a Notch 1-induced leukemia model. Blood Aug 3. [Epub ahead of print]

Qiu J, Takagi Y, Harada J, et al. 2009. p27Kip1 constrains proliferation of neural progenitor cells in adult brain under homeostatic and ischemic conditions. Stem Cells 27: 920-927.

Yu H, Yuan Y, Shen H, Cheng T. 2006. Hematopoietic stem cell exhaustion impacted by p18 and p21in opposite manners. Blood 107: 1022-1206. Full Text

Yuan Y, Shen H, Franklin DS, et al. 2004. In vivo self-renewing divisions of hematopoietic stem cells in vivo are increased in the absence of the early G1-phase inhibitor, p18INK4c. Nat. Cell Biol. 6: 436-442.

Yuan Y, Yu H, Boyer MJ, et al. 2006. Hematopoietic stem cells are not the direct target of spontaneous leukemic transformation in p18(INK4C)-null reconstituted mice. Cancer Res. 66: 343-351. Full Text

Michael Sheetz

Cai Y, Sheetz MP. 2009. Force propagation across cells: mechanical coherence of dynamic cytoskeletons. Curr. Opin. Cell Biol. 21: 47-50.

Engler AJ, Sen S, Sweeney HL, Discher DE. 2006. Matrix elasticity directs stem cell lineage specification. Cell 126: 677-689. Full Text

Giannone G, Dubin-Thaler BJ, Rossier O, et al. 2007. Lamellipodial actin mechanically links myosin activity with adhesion-site formation. Cell 128: 561-575. Full Text

Kostic A, Lynch CD, Sheetz MP. 2009. Differential matrix rigidity response in breast cancer cell lines correlates with the tissue tropism. PLoS One 4(7): e6361 Full Text

Moore SW, Biais N, Sheetz MP. 2009. Traction on immobilized netrin-1 is sufficient to reorient axons. Science 325: 166.

Puklin-Faucher E, Sheetz MP. 2009. The mechanical integrin cycle. J. Cell. Sci.122: 179-186. Full Text

Sawada Y, Tamada M, Dubin-Thaler BJ, et al. 2006. Force sensing by mechanicalextension of the Src family kinase substrate p130Cas. Cell 127: 1015-1026. Full Text

Vogel V, Sheetz MP. 2009. Cell fate regulation by coupling mechanical cycles to biochemical signaling pathways. Curr. Opin. Cell Biol. 21: 38-46.

Ibrahim Domian

Bu L, Jiang X, Martin-Puig S, et al. 2009. Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature 460: 113-117.

Chien KR, Domian IJ, Parker KK. 2008. Cardiogenesis and the complex biology of regenerative cardiovascular medicine. Science 322: 1494-1497.

Ulrich Martin

Blömer U, Gruh I, Witschel H, Haverich A, Martin U. 2005. Shuttle of lentiviral vectors via transplanted cells in vivo. Gene Ther. 12: 67-74.

Gruh I, Wunderlich S, Winkler M, et al. 2008. Human CMV immediate-early enhancer: a useful tool to enhance cell-type-specific expression from lentiviral vectors. J Gene Med. 10: 21-32.

Gruh I, Beilner J, Blomer U, et al. 2006. No evidence of transdifferentiation of human endothelial progenitor cells into cardiomyocytes after coculture with neonatal rat cardiomyocytes. Circulation 113: 1326-1334. Full Text

Mauritz C, Schwanke K, Reppel M, et al. 2008. Generation of functional murine cardiac myocytes from induced pluripotent stem cells. Circulation 118: 507-517. Full Text

Schwanke K, Wunderlich S, Reppel M, et al. 2006. Generation and characterization of functional cardiomyocytes from rhesus monkey embryonic stem cells. Stem Cells 24: 1423-1432. Full Text

Winkler ME, Mauritz C, Groos S, et al. 2008. Serum-free differentiation of murine embryonic stem cells into alveolar type II epithelial cells. Cloning Stem Cells 10: 49-64.

Wunderlich S, Gruh I, Winkler ME, et al. 2008. Type II pneumocyte-restricted green fluorescent protein expression after lentiviral transduction of lung epithelial cells. Hum. Gene Ther.19: 39-52.

Yong Zhao

Zhao Y, Li XC. 2007. Transplant tolerance: is it really free of concerns? Trends Immunol. 28: 376-377.

Zhao Y. 2007 The different effects of cyclosporin A and rapamycin on regulatory CD4+CD25+ T cells: potential relationship with transplant tolerance induction. Expert Review of Clinical Immunol. 3: 254-259. Full Text

Yi H, Zhen Y, Zeng C, et al. 2008. Depleting anti-CD4 monoclonal antibody (GK1.5) treatment: influence on regulatory CD4+CD25+Foxp3+ T cells in mice. Transplantation 85: 1167-1174.

Wang H, Zhao L, Sun Z, et al. 2006. The potential side effects of Cyclosporin A: its inhibition on CD4+CD25+ regulatory T cells in mice. Transplantation 82: 1484-1492.

Zhang A, Qu Y, Zhang B, Zhang et al. 2007. The different effects of indirubin on effector and CD4+CD25+ regulatory T cells in mice:potential implication for the treatment of autoimmune diseases. J. Mol. Med. 85: 1263-1270.

Zeng C, Zhen Y, Shu SA, et al. 2007. Inhibition of B and T lymphocyte attenuator expression by immunosuppressive drug cyclosporin A. J. Autoimmunity 28: 234-244.

Hongkui Deng

Jiang W, Shi Y, Zhao D, et al. 2007. In vitro derivation of functional insulin-producing cells from human embryonic stem cells. Cell Res.17: 333-344.

Li L, Li F, Qi H, et al. 2008. Coexpression of Pdx1 and betacellulin in mesenchymal stem cells could promote the differentiation of nestin-positive epithelium-like progenitors and pancreatic islet-like spheroids. Stem Cells Dev.17: 815-823.

Zhang D, Jiang W, Liu M, et al. 2009. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res.19: 429-438.

Robert Chunhua Zhao

Chen L, Zhang W, Yue H, et al. 2007. Effects of human mesenchymal stem cells on the differentiation of dendritic cells from CD34+ cells. Stem Cells Dev.16: 719-731.

Liao L, Li L, Zhao RC. 2007. Stem cell research in China. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 362: 1107-1112. Full Text

Liu Y, Yan X, Sun Z, et al. 2007. Flk-1+ adipose-derived mesenchymal stem cells differentiate into skeletal muscle satellite cells and ameliorate muscular dystrophy in mdx mice. Stem Cells Dev.16: 695-706.

Yan X, Liu Y, Han Q, et al. 2007. Injured microenvironment directly guides the differentiation of engrafted Flk-1(+) mesenchymal stem cell in lung. Exp. Hematol. 35: 1466-1475.

Zhang B, Liu R, Shi D, et al. 2009. Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2-dependent regulatory dendritic cell population. Blood 113: 46-57.

James Yoo

Atala A, Bauer SB, Soker S, et al. 2006. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet 367: 1241-1246.

Eberli D, Freitas Filho L, Atala A, Yoo JJ. 2009. Composite scaffolds for the engineering of hollow organs and tissues. Methods. 47: 109-115.

Kim B, Atala A, Yoo JJ. 2008. A collagen matrix derived from bladder can be used to engineer smooth muscle tissue. World J Urol. 26: 307-314.

Oh SH, Ward CL, Atala A, et al. 2009. Oxygen generating scaffolds for enhancing engineered tissue survival. Biomaterials. 30: 757-762.

Xu T, Olson J, Zhao W, et al. 2008. Characterization of cell constructs generated with inkjet printing technology using in vivo magnetic resonance imaging. J. Manuf. Sci. Eng. 130: 021013.

Xu T, Rohozinski J, Zhao W, et al. 2008. Inkjet mediated gene transfection into living cells combined with targeted delivery. Tissue Eng. Part A. 15: 95-101.

Yilin Cao and Wei Liu

Cao D, Liu W, Wei X, et al. 2006. In vitro tendon engineering with avian tenocytes and polyglycolic acids: a preliminary report. Tissue Eng. 12: 1369-1377.

Cen L, Liu W, Cui L, et al. 2008. Collagen tissue engineering: development of novel biomaterials and applications. Pediatr. Res. 63: 492-496.

Cui L, Wu Y, Cen L, et al. 2009. Repair of articular cartilage defect in non-weight bearing areas using adipose derived stem cells loaded polyglycolic acid mesh. Biomaterials 30: 2683-2693.

Cui L, Liu B, Liu G, et al. 2007. Repair of cranial bone defects with adipose derived stem cells and coral scaffold in a canine model. Biomaterials 28: 5477-5486.

Luo X, Zhou G, Liu W, et al. 2009. In vitro precultivation alleviates post-implantation inflammation and enhances development of tissue-engineered tubular cartilage. Biomed Mater.4: 25006.

Yan D, Zhou G, Zhou X, et al. 2009. The impact of low levels of collagen IX and pyridinoline on the mechanical properties of in vitro engineered cartilage. Biomaterials 30: 814-821.

Zhao G, Yin S, Liu G, et al. 2009. In vitro engineering of fibrocartilage using CDMP1 induced dermal fibroblasts and polyglycolide. Biomaterials 30: 3241-3250.

Zhou G, Liu W, Cui L, et al. 2006. Repair of porcine articular osteochondral defects in non-weightbearing areas with autologous bone marrow stromal cells. Tissue Eng. 12: 3209-3221.

Haiyan Xu

Han Z, Kong H, Meng J, et al. 2009. Electrospun aligned nanofibrous scaffold of carbon nanotubes-polyurethane composite for endothelial cells. J. Nanosci. Nanotechnol. 9: 1400-1402.

Meng J, Song L, Xu H, et al. 2005. Effects of single-walled carbon nanotubes on the functions of plasma proteins and potentials in vascular prostheses. Nanomedicine 1: 136-142.

Ning Gu

Ge Y, Zhang Y, Xia J, et al. 2009. Effect of surface charge and agglomerate degree of magnetic iron oxide nanoparticles on KB cellular uptake in vitro. Colloids Surf B Biointerfaces. [Epub ahead of print]

Ge Y, Zhang S, He S, et al. 2009. Fabrication and characterization of chitosan-poly(acrylic acid) magnetic nanospheres. J. Nanosci. Nanotechnol. 9: 1287-1290.

Sun JF, Mao YQ, Guo ZR, et al. 2009. Time-varied magnetic-field induced monolayer formation and re-aggregation of Au nanoparticles during solvent evaporation. J. Nanosci Nanotechnol. 9: 1156-1159.

Xia JG, Zhang S, Zhang Y, et al. 2009. Maghemite nanoparticles and their protamine derivatives: cellular internalization and effects on cell-cycle progress. J Nanosci Nanotechnol. 9: 1025-1028.

Yang F, Li Y, Chen Z, et al. 2009. Superparamagnetic iron oxide nanoparticle-embedded encapsulated microbubbles as dual contrast agents of magnetic resonance and ultrasound imaging. Biomaterials. 30: 3882-3890.

Sponsorship

Silver:

Academy Friends:

Deutsche Forschungsgemeinschaft

Medical Research Council

Novartis

Pfizer

How can scientists improve the homing and engraftment of progenitor or stem cells at sites of tissue damage?

Will the use of cardiac stem cells improve results of cell therapy for cardiovascular disease?

Can boosting thymic function induce tolerance to foreign cells?

Which immune cell type is the best target for inducing tolerance?

Will the state of tolerance be reversed by some types of infectious disease?

Can local immune privilege be induced in all transplanted organs?

How can memory T and B cell responses be controlled?

Will tissues repair themselves after autoimmunity is controlled?

How does IL-1Ra protect cells against non cell cycle-specific drugs?

How does the presence of leukemic cells change the stem cell niche?

How do leukemic cells alter the levels of expression of cell cycle genes in normal hematopoietic stem cells?

How is input about the mechanical environment translated into growth and differentiation cues in metastatic breast cancer cells and mesenchymal stem cells?

Can cardiomyocytes or other cell types be cultivated in large enough numbers for use in cell therapies?

Is injection of precursor cells sufficient to repair heart or other organ damage?

How can stem cells be guided to form complex tissues?

What is the molecular mechanism of Flk1+ differentiation into hematopoietic cells?

How can neural connections be restored after severe damage to a tissue?

What is the best source of cells for tendon, cartilage, and bone repair?

Can a composite material mimic the basement membrane in vascular grafts?

Will pig islet cells and livers function well enough to replace human organs?

Will a new paradigm arise to finance research that is not likely to generate windfall profits for investors or companies?

Speaker:
Michael Sheetz, Columbia University, National University of Singapore

Highlights

• Mechanical forces influence cell migration, growth, and basic signal transduction processes.
• Breast cancer cells that metastasized to bone grow better on a hard medium than those cells that metastasized to the lung. The converse is true on soft media.
• The rigidity of the substrate affects the differentiation of mesenchymal stem cells.
• Stretching of the cytoskeleton induces the binding of proteins at focal adhesions.
• Stretching of the cytoskeletal protein p130Cas greatly increases phosphorylation of the protein.

Mechanical forces affect cell behavior

Michael Sheetz of Columbia University and the National University of Singapore is studying mechanical forces that enable cells to migrate, organize subcellular structures such as collagen, and transduce signals that determine cell fate. Although mechanical forces are central to the basic operation of organisms, study of these forces has been neglected while scientists focused on biochemical aspects of cell and tissue function.

To illustrate the importance of mechanical forces, Sheetz showed data of metastatic breast cancer cell growth on either hard or soft media. The team first grew human breast cancer cells in immunodeficient “nude” mice and transferred them to new mice until the cells metastasized to bone or to the lung. They isolated the metastatic cells and repeated the procedure in a new set of mice. Finally, they investigated how well the cells grew on hard versus soft fibronectin media.

Cells that had metastasized to bone grew better on a hard medium whereas those that had targeted to the lung grew better on soft media, indicating that the physical/mechanical environment plays a role in determining whether a cell will grow or remain quiescent. Additional evidence that the physical environment affects cell behavior is evident in work by Engler and others, which showed that mesenchymal stem cells differentiated into different cell types depending on the rigidity of the substrate upon which they were grown, said Sheetz.

Coordination of subcellular modules for cell motility

Sheetz’s lab is working to understand the mechanics of subcellular modules in the 20-1000 nm range. They plan to fully describe the 20-30 motile functions of a cell by defining the steps involved in a mechanochemical function, describing the physical and biochemical basis of each step, and developing a quantitative model of each step and of the overall function.

Sheetz described a part of this work elucidating the mechanical process by which a cell can migrate along a surface. Using a combination of microscopy techniques, they were able to determine the interaction of actin filaments with myosin and the adhesion site in a migrating cell. First, two layers of actin develop by polymerization at the leading edge. When the upper layer interacts with myosin it gets pulled away from the leading edge and condensed at the back in radial actin arrays. Then new actin starts to polymerize at the leading edge as well as at the adhesion. The actin then moves back and gets pulled by a new myosin and the cycle continues, explained Sheetz. What is significant about this work is that it sheds light on a mechanical process that can’t be described purely by a biochemical model, he emphasized.

Translation of mechanical stimuli to biochemical signals

Sheetz enumerated a number of other situations in which force is a cofactor in biochemical reactions, including motor protein velocity, stretch-sensitive ion channel signaling, and cytoskeletal protein stretching. His team has shown that stretching the cytoskeleton leads to binding of many proteins at certain sites called focal adhesions. These proteins disperse when the tension is released.

Sheetz’s group investigated the effects of stretching on p130Cas, a protein localized to the cytoskeleton and involved in cell migration, survival, transformation, and invasion. They devised a method to stretch p130Cas in vitro, using antibodies to attach the protein to a stretchable surface in the presence of active Src kinase, which activates the protein by phosphorylation. They found that phosphorylation of the protein was dependent upon stretching of the substrate domain. Unfolding of the domain by stretch exposes the phosphorylation sites, leading to a seven-fold increase in phosphorylation of the protein. Thus, mechanical force on the cytoskeleton is translated to a biochemical signal within the cell. Understanding the effects of mechanical forces on proteins and cells is critical to a complete characterization of their behavior, whether for regenerative medicine or other clinical applications.

Speakers:
Wei Han, Shanghai Jiao Tong University
Tao Cheng, Chinese Academy of Medical Sciences

Highlights

• Introducing the cytokine antagonist IL-1Ra may improve the speed and quality of bone marrow regeneration after chemotherapy.
• IL-1Ra drives stem cells and progenitor cells into G0.
• IL-1Ra appears to boost bone marrow cells after treatment with either cell cycle- or non-cell cycle-specific chemotherapeutic agents.
• The level of normal hematopoietic stem cells is depressed after one month in a leukemic environment.
• In this environment, cell cycle regulators keep HSCs in a quiescent state and block the further maturation needed to replenish blood cells.
• The repopulation advantage of the HSCs from the leukemic environment in new recipients is associated with the altered expression of several genes that have been shown to be important for HSC cell cycle control and self-renewal.

Regeneration of blood cells after chemotherapy

Wei Han and his group at Shanghai Jiao Tong University are trying to understand the natural regenerative capacity of the body with the aim of developing more effective therapeutics. This natural capacity is evident during “holidays” from radiation and chemotherapy for cancer, when the toxic regimen is temporarily halted to allow bone marrow cells to recover. Unfortunately, regeneration of normal cells is accompanied by regrowth of the cancer cells, and over time the body’s ability to recover is weakened. One goal of Han’s group is to find the molecules that control the speed and quality of bone marrow regeneration after chemotherapy, reducing the holiday time required for the treatment.

Han pointed out that after chemotherapy, blood cell numbers return to normal, but never exceed the initial level, indicating that a homeostatic mechanism is at work. So his group used gene expression technology after 5-FU chemotherapeutic treatment to screen for the positive and negative regulators of bone marrow regeneration that restore balance to the system. One intriguing gene that came out of the screen was the cytokine antagonist IL-1Ra, which is upregulated immediately after treatment.

IL-1 is a cytokine that is secreted from monocytes and macrophages and has a wide variety of effects, including the induction of inflammation. The antagonist, IL-1Ra, binds to the same receptor as IL-1, preventing an IL-1 response. To understand the role of IL-1Ra in bone marrow regeneration, Han’s team transiently overexpressed the protein in murine muscle. They found that overexpression of the antagonist caused a decrease in the levels of blood cells in the periphery, but an increase in the bone marrow. Injection of recombinant IL-1Ra in mice before chemotherapy increased the survival rate of the animals by promoting bone marrow recovery, even after two or three rounds of chemotherapy.

Han proposed that the mechanism by which the IL-1Ra treatment works is by driving bone marrow cells out of the cell cycle, rendering them quiescent and resistant to chemotherapeutic drugs. The group’s data show that a greater proportion of bone marrow stem and progenitor cells are in G0 in the IL-1Ra treated mice than in control mice. Interestingly, the group found similar protective effects when they tested IL-1Ra on animals treated with non cell cycle-specific chemotherapeutic drugs, such as cyclophosphamide. Again, these effects were sustained after two or three rounds of chemotherapy.

But what happens to the cancer cells? If they are also moved out of the cell cycle, the treatment wouldn’t work well. Early experiments indicate that IL-1Ra does not drive cancer cells out of the cell cycle, presumably because their cell cycle regulation is damaged, said Han. Some additional evidence to support this hypothesis comes from Amgen, a company that has marketed IL-1Ra to treat patients with rheumatoid arthritis (RA). They have found that RA patients that also have cancer do not have worse outcomes than other cancer patients, suggesting that the drug does not have the same cell cycle effect on cancer cells.

Effects of the disease environment on stem cells

By their very nature, stem cells hold the potential to regenerate damaged tissue and to cause cancer. Researchers are trying to understand what differentiates stem cells from cancer cells and to define the limitations on stem cell growth. Tao Cheng of the Institute of Hematology at the Chinese Academy of Medical Sciences is investigating the factors that regulate the growth and survival of normal hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) in the same niche. “How diseased conditions impact normal tissue stem cells is critical for the success of stem cell transplants in recipients and yet it is largely unknown,” said Cheng.

Researchers can generate a model of leukemia by overexpressing the Notch1 gene in hematopoietic cells and injecting them into mice whose blood cells have been destroyed by radiation. To examine the effects of the leukemic environment on normal hematopoietic stem cells, Cheng’s team co-injected Notch-transformed cells and normal HSCs into the irradiated mice and compared the stem cells levels over time to those in control mice. One week after transplantation they saw an increase in hematopoietic progenitor cells (HPCs), a more committed state, relative to control mice. But after one month, they found a fivefold decrease in the levels of HSCs and HPCs in the leukemic mice compared to control mice. The number of cells that a single stem cell could generate was also fivefold less in the leukemic mice. In two assays to assess stem cell function, the frequency of colony-forming cells and cobblestone area-forming cells was decreased.

Interestingly, when stem cells were transplanted from leukemic mice into a normal secondary recipient along with normal stem cells from control mice, the stem cells from leukemic mice functioned better than the control cells, showing about a 10-fold difference in levels in the bone marrow. This result suggests that the suppressive effect of the leukemic environment is reversible, said Cheng.

There is an increased fraction of quiescent stem cells in the leukemic marrow at week 2.

To determine the molecular basis of their results, Cheng’s team examined a number of factors that affect the cell cycle. Earlier work had shown that in normal stem cells, the cell cycle regulator p21 kept cells out of the cell cycle, p27 blocked differentiation at a later stage, and p18 blocked the self-renewal pathway of a cycling cell, driving it toward differentiation or apoptosis. Cheng found that in a leukemic environment, the expression of p18 in hematopoietic stem cells was significantly decreased, whereas p21 was increased, keeping the cells in a quiescent state and inhibiting maturation. There was no statistical difference in p27 expression between the cells from leukemic and control marrow. Among the self-renewal associated genes, the expression of Gfi1, c-Myc, and Notch1 was significantly increased.

From these results, Cheng proposed that although HPCs may rapidly proliferate in the early stage of leukemia, the production of mature blood cells is ultimately limited due to the exhaustion of HPC pools that cannot be replenished from the more quiescent HSCs in the late stage of leukemia development.

Speakers:
Gerald Chan, Morningside Group
Brock Reeve, Harvard Stem Cell Institute
Leonide Saad, Proteus Venture Partners
Leon Chen, Fidelity Asia Ventures
Alex Fowkes, Pfizer (China) Research and Development

Highlights

• Public-private partnerships may finance more research that traditional companies view as too risky to tackle.
• Venture capital may begin to invest in the regenerative medicine arena now that the field has matured.
• Large organizations that can invest for the long term are more likely to fund regenerative medicine than others.
• A strong regulatory framework is required to ensure that negative results don’t set back the field.

Who should pay?

Like most pharmaceutical research, regenerative medicine research is expensive, but unlike pharma, the end product is a technique or medical procedure, not a pill or medical device. So who is going to pay for it if the returns on investment are uncertain? Gerald Chan, cofounder of Morningside Group, an investment firm engaged in private equity and venture capital investments, posed this question to a distinguished group of panelists representing public and private organizations interested in regenerative medicine.

Brock Reeve, executive director of the Harvard Stem Cell Institute, suggested that public-private partnerships that finance individual projects are replacing the traditional approach in which a biotech company is founded to commercialize a basic research finding. Venture capital is not enthusiastic about funding this work because it is too speculative and may not show results in the five years, the typical mark at which the funds expect to see something. Private foundations in the United States like the Myelin Repair Foundation are rounding up a handful of academic labs, raising money, and working with the commercial sector to move things into the market with goal-driven deliverables. So a hybrid nonprofit sector is arising to tackle the problem, said Reeve.

Leonide Saad, vice president of Proteus Venture Partners, a venture capital fund focused entirely on regenerative medicine concurred that the field presents difficulties to the traditional VC model. There hasn’t been a single commercial success in the field yet, and even development of a successful therapy like bone marrow transplant doesn’t translate to a financial windfall for anyone involved. Any researcher looking to a venture capital group for funding needs to have a solid business model, intellectual property rights, an understanding of the regulatory path involved, and a team with a track record in building companies. As a researcher, it is important to seek out a venture capital group that has expertise in your area.

Leon Chen of Fidelity Asia Ventures described how his group, which is 100% internally funded, has the ability to invest in a company and stick with it over the long term, thereby eliminating the need to see quick results. The fund examines trends and looks for areas that are going to make a major impact on the country, such as the Internet and health care. Venture capital will play an important role in the future, but right now the government has made the biggest impact on the field of regenerative medicine in China by investing heavily in the life sciences and biotech infrastructure, said Chen.

Alex Fowkes, executive director of Pfizer (China) Research and Development noted that pharmaceutical companies are waiting until much further along in the process before funding research, funding clinical trials rather than preclinical research, for example. Preclinical research funding is designed to feed the clinical pipeline and a lot of the investment goes to modalities that are validated, such as small molecules. But new approaches such as RNAi and regenerative medicine are the focus of small research units. Pfizer’s R&D is not investing heavily in stem cell therapy yet, Fowkes said.

Will investing in regenerative medicine research ever be a money-making endeavor?

Gerald Chan returned to the question of whether it was fundamentally contradictory to invest in regenerative medicine with the goal of making money. Perhaps it should be left to government or to the nonprofit sector, he posited. He pointed out the fact, documented in the book Science Business: The Promise, the Reality, and the Future of Biotech, that over the past 30 years, more money has been lost investing in biotech than gained.

But the panelists weren’t ready to concede defeat. Leonide Saad argued that the tremendous scientific progress that has been made since the 1970s laid a strong foundation so that new breakthroughs now occur at a much faster rate. The regenerative medicine industry went through the same growth and bubble burst as the Internet, he said. But when the private sector refused to finance the research afterwards, the government stepped in—particularly some state governments—pouring money into stem cell research from 2002 to 2007. Now the field has matured to the point where some companies are viable, Saad said. Brock Reeve argued that a new business model in which an investor can find intellectual property in one place, find a project manager (CEO), and outsource everything else, allows for flexibility that didn’t exist a few years ago because that dispersion of resources didn’t exist.

The nonprofit sector has its own difficulties, Brock argued. Accelerator funds provided by universities to help drive research products further toward commercialization are often inadequate to get the job done. Foundations are driven to find the "homerun" instead of funding useful projects with a more limited scope. They are also in competition with each other, when a more efficient use of time and capital would come from pooling resources, he said.

Chan turned the focus of the panelists to the role of government regulation in regenerative medicine. Unlike in the U.S. and Europe, China does not have strict regulations in this area and stem cell therapies are currently being administered in its hospitals. The panelists generally agreed that regulations are important for minimizing adverse effects in patients. When something goes wrong, as when a young patient died in a gene therapy trial in the U.S., there is a backlash that effectively kills the funding research in that area.

Speaker:
Stefanie Dimmeler, University of Frankfurt

Highlights

• Bone marrow mononuclear cells can improve survival outcomes in acute myocardial infarction patients.
• Short-term exposure to mononuclear cells does not account for the improvements in a mouse model.
• Mononuclear cells contribute to the vascular lineage when injected into damaged murine hearts.

Vascularization in cardiac regeneration

Used primarily for reconstituting the immune system after chemotherapy to treat leukemia, bone marrow is the only source of stem cells that has a record of success in the clinic. Given the multipotent nature of bone marrow cells and the incomplete understanding of their regenerative capacities, researchers have been investigating whether these cells can regenerate other tissues as well.

Injecting bone marrow mononuclear cells following acute myocardial infarction shows good early results.

Stefanie Dimmeler of the University of Frankfurt described the results of a randomized, double-blind, placebo-controlled study investigating the regenerative capacity of bone marrow mononuclear cells in 200 patients with acute myocardial infarction. After infarction, bone marrow was taken from the patients, mononuclear cells were purified away from other bone cells, and then 230 million cells were injected via a balloon catheter into the damaged region of the heart.

The study showed that in patients who received the cell therapy there was a significant increase in ejection fraction (the percentage of blood pumped out of the ventricles with each beat). Remodeling of the heart, which leads to heart failure, also appeared to be prevented. The therapy improved the survival of the patients as well—after two years, 95% of the treated patients had event-free survival compared to 86% of control patients. Dimmeler noted that this was a small study and that greater numbers would be required to confirm the findings.

The search for the mechanism

A number of mechanisms may account for the healing effects of bone marrow cells after myocardial infarction, explained Dimmeler. It is possible that the bone marrow cells differentiate into cells required for vasculogenesis and/or cardiomyogenesis. In addition, or perhaps alternatively, paracrine effects—growth factor and cytokine release from the cells—may promote the genesis of new blood vessels, prevent apoptosis of cardiomyocytes, and modulate inflammation. Understanding the mechanism by which the injected cells contribute to healing is important because it affects the route researchers take to improve the therapy. For example, if paracrine effects dominate, it might be possible to provide growth factors and cytokines to the damaged area directly instead of injecting cells. However, if the cells need to be incorporated into the tissue for benefits to be seen, techniques that improve the rate of engraftment will be critical. Techniques that drive cells toward a particular differentiated state may be used if the repair can be attributed to that cell type.

Dimmeler’s team was interested in distinguishing between short-term paracrine effects versus the long-term effects that would be seen if the cells were incorporated into existing tissue. To tackle this question, her team employed a genetic technique that would enable them to initiate the death of injected bone marrow cells several weeks after treatment. Because the thymidine kinase (TK) gene product turns the prodrug gancyclovir into a toxic product, transduction of a cell with that gene renders it susceptible to gancyclovir treatment. Dimmeler’s group used a lentivirus vector to transduce bone marrow cells with either the TK gene or the green fluorescent protein (GFP) gene.

They induced acute myocardial infarction in mice and injected bone marrow cells carrying either the TK gene or the GFP control. After two weeks, they injected gancyclovir to kill the cells carrying the TK gene. Destroying the cells had a negative effect on the efficacy of the therapy, eliminating the improvement of cardiac function and neovascularization that occurs in the control. Thus, the short-term effects were not sufficient to account for the therapeutic value of the treatment.

Animals in which transplanted bone marrow cells are depleted two weeks after injection have worse outcomes than control animals.

Since several different cell types could contribute to the improvement in heart function, Dimmeler next wanted to determine the final differentiation state of the bone marrow cells. The group created constructs in which the thymidine kinase gene was driven by cell type-specific promoters so that they could selectively kill endothelial cells, smooth muscle and fibroblast cells, or cardiomyocytes. They repeated the experiment described above and found that depleting all the cells had a greater effect on survival (more animals died) than depleting any one cell type.

Dimmeler noted that to improve the treatment, the engraftment of cells must be enhanced. Less than 5% of the injected cells home to the heart, she said. At the same time, the cells taken from an infarct patient may not be that healthy. The team is investigating whether nitric oxide synthase pretreatment of cells could enhance homing and cellular function. Other ideas are to pretreat the damaged area with peptide nanofibers, growth factors, or low energy shockwave therapy to activate the tissue by stimulating release of paracrine factors.

Speaker:
Anita Chong, University of Chicago

Highlights

• Any attempt to transplant foreign cells or tissues into the body must be accompanied by a method to induce the immune response either through pharmacological immunosuppression or through the induction of tolerance.
• Tolerance of foreign antigens could be induced if the antigens gained access to the thymus, where T cells that recognize them could be eliminated.
• A greater understanding of the role of regulatory T cells and tolerogenic dendritic cells may allow researchers to induce peripheral tolerance.
• Several ligands that induce local tolerance, including PDL-1, have been identified.
• In model systems, β-cells can spontaneously regenerate if the autoimmune response in type 1 diabetes is controlled.

Immune tolerance

Foreign cells transplanted into a person for therapeutic purposes will be rejected by the recipient’s immune system unless immunosuppressive measures are taken. In instances like type 1 diabetes, tissue needs replacement in the first place because an autoimmune response destroyed it, while replacing the tissue, even with the recipient’s own cells, will result in the eventual destruction of the new tissue as well.

Any graft therapy must be accompanied by a treatment that modulates the immune system response.

For the field of regenerative medicine, this means that any graft therapy will need to be accompanied by a treatment that modulates the immune system response. In Beijing, Anita Chong of the University of Chicago described the state of the field of immune tolerance.

Antigens recognized by the T cells that mediate graft rejection are referred to as HLA antigens. Because these antigens are so polymorphic, it is not usually possible to find a perfect donor match, Chong explained. Suppressing the immune system with drugs is a suboptimal approach that leaves the patient susceptible to infections and tumors.

A better, though more challenging, option is to harness the body’s natural mechanism for inducing tolerance to antigens. In the developing immune system, T cells are subjected to selection in the thymus based on their reaction to ligands on epithelial cells and bone-marrow derived cells (thymocytes, macrophages, and dendritic cells). This is a complex process, but in short, thymocytes that have a strong affinity to the ligands on the epithelial cells—and thus would generate an autoimmune response—are deleted, whereas thymocytes that have a low affinity for these ligands are selected for survival and differentiation into mature T cells. This process is dependent on both epithelial and bone marrow-derived cells.

Central tolerance is induced in the thymus, where T cells that bind strongly to "self" ligands will be negatively selected.

Tolerance to foreign antigens could be induced if the antigens gained access to the thymus, where T cells that recognize them could be eliminated. But the thymus atrophies rapidly over time. Efforts are currently underway to boost thymic function or to transplant thymic tissue or bone marrow to induce tolerance to donor cells, said Chong.

Peripheral tolerance

Another important part of the immune response is peripheral tolerance, which occurs after T and B cells mature and enter the circulation. One way to induce tolerance to foreign antigens is to globally deplete all T and B cells, but this leaves the patient vulnerable to infection. Another approach is to block the activation of T cells. These cells require two signals to become activated and differentiate into effector cells. The first signal is through the T cell receptor (TCR), which detects the antigen, and the second is from one of a series of co-stimulatory molecules. T cells receiving the first signal without the second will be inactivated. Thus, reagents that block co-stimulatory molecules have been developed with the goal of inducing tolerance, said Chong. This approach has had some success but doesn’t lead to permanent tolerance.

A better understanding of Treg cells and tolerogenic dendritic cells may lead to strategies for inducing immune tolerance.

Investigations into the mechanism of peripheral tolerance indicated that, in addition to the selective inactivation and deletion of antigen-specific T cells, there was also induction of a class of immunomodulatory T cells called regulatory T cells (Tregs). These antigen-specific cells are thought to control peripheral immune responses by producing inhibitory cytokines, killing effector cells, and targeting another class of cells called dendritic cells, which present antigens to T cells.

Recently a class of dendritic cells has been identified that promotes the establishment of peripheral tolerance. These tolerogenic dendritic cells are immature or alternatively activated cells that are unresponsive to “danger” signals, produce suppressive cytokines, and express high levels of inhibitory molecules. "The ability to regenerate large numbers of tolerogenic dendritic cells," Chong explained, “may be a way in which regenerative medicine can contribute to the development of peripheral tolerance."

Other methods of inducing tolerance

One intriguing model of tolerance is local tissue-specific tolerance, known as immune privilege, found in the placenta, brain, testes, and the anterior chamber of the eye. Molecular studies have identified a number of ligands thought to induce peripheral tolerance at these sites. One of these, called PDL-1 is expressed in the parenchyma, including on endothelial cells in the lung and β-cells in the pancreas, as well in the hematopoietic system, said Chong. When PDL-1 binds the PD1 receptor on T cells, it inactivates the T cell, thereby inducing tolerance. In the future, these molecules may be introduced into cells by genetic engineering to induce local tolerance, she suggested.

Chong described the numerous barriers that remain to inducing tolerance in the clinic. Stimulating the innate immune response can block the induction of tolerance and even reverse established tolerance. Another big barrier to inducing tolerance is the presence of memory T and B cells. Memory immune response kinetics are more rapid and less easily controlled by reagents that are effective at controlling the immune response during the system’s first encounter with a foreign antigen. A number of laboratories are working to identify the signals that restrict memory T cell responses and to find new targets for intervention, she said.

Chong concluded by pointing out that blocking the autoimmune response that leads to the destruction of β-cells in type 1 diabetes could enable the body to regenerate these cells on its own, obviating the need for transplants altogether. Several groups observed spontaneous islet regeneration from endogenous β-cells when the autoimmune response was controlled.

Speakers:
Yong Zhao, Chinese Academy of Sciences
Hongkui Deng, Peking University
Robert Chunhua Zhao, Chinese Academy of Medical Sciences

Highlights

• Cross tolerance may potentially create obstacles to using tolerance to induce graft acceptance.
• Immunosuppressive drugs have different effects on effector and regulatory T cells.
• Demethylation of the Foxp3 transcription factor can boost levels of regulatory T cells.
• A stepwise approach to stem cell maturation can yield mature, insulin-producing β-cells.
• Flk1+ multipotent stem cells have regenerative capacity and immunomodulatory functions.

Immunosuppression and transplantation tolerance

Cross reactivity in antigen recognition by the T cell receptor is essential to promote an efficient and diverse immune response. The existence of cross-reactive T cells implies that cross tolerance is also a possibility; that is, induction of tolerance to one antigen by neutralizing a cross-reactive T cell will simultaneously induce tolerance to any other antigen to which the cell responded. Thus, researchers interested in inducing tolerance to prevent graft rejection should consider the possibility that inducing tolerance to donor antigens may also neutralize the response to dangerous pathogens, cautioned Yong Zhao of the Institute of Zoology at CAMS.

The immune response that determines whether a graft will be rejected is mainly mediated by a balance of regulatory T (Treg) cells and effector T cells. As Anita Chong mentioned, patients who receive organ transplants must take immunosuppressive drugs for the rest of their lives. It is known that these drugs work by decreasing the number of effector cells in the patient’s blood, but what happens to the Treg cells, the T cell population that suppresses activation of the immune system?

To find out, Zhao and his team treated mice with different immunosuppressive drugs and looked at the effects on the levels and function of Treg cells. They found strikingly different responses depending upon which drug they used. For example, cyclosporine A significantly decreased the levels of Treg cells in the spleen and blood, whereas rapamycin relatively increased the levels of Treg cells in the spleen. Zhao suggested that immunosuppressive drugs could be classified based on their effects on Treg cells; the class that suppresses effector cells but boosts Treg cells would be appropriate for inducing tolerance while preventing the development of an autoimmune response.

Rapamycin enhanced Treg levels in the spleens of mice treated with the drug.

Though the function of Treg cells is still poorly understood, it is clear that having normal levels of these cells is important for preventing autoimmune reactions. One possible way to boost Tregs would be to remove inhibitory methylation from the promoters of the transcription factor Foxp3, which is required for Treg development and function. Zhao’s group found that the demethylation drug, 5-Aza-2’-deoxycitidine (DAC), which is used to treat cancer, enhanced Treg cell development in the thymus. They went on to show that DAC treatment inhibited the occurrence of diabetes in NOD mice, a strain of mice that spontaneously develops an autoimmune-based diabetes.

Zhao’s group is also working on the molecular basis of T cell function. B and T cell lymphocyte attenuator (BTLA) is a cell surface protein that is an important negative regulator of T-cell activation. Mice lacking the protein are more susceptible to autoimmune disease, said Zhao. Looking again at the effects of immunosuppressive drugs, Zhao found that cyclosporine treatment but not rapamycin treatment significantly inhibits BTLA expression on CD4+ T cells, suggesting that rapamycin might have a less destructive effect on negative regulation of the immune system via this receptor.

Stem cell therapy for type 1 diabetes

While some researchers are trying to understand how the immune system avoids attacking “self,” others are tackling the consequences of such an attack. Type 1 diabetes is caused by destruction of the β-cells of the Islets of Langerhans, regions of the pancreas that produce insulin. Current therapy for type 1 diabetes consists of insulin injection, pancreas transplantation, or islet transplantation. There are severe limitations to the latter two therapies, however. As in all cases of transplantation, donors are not readily available and the immunosuppressive regimen can be a problem.

Hongkui Deng's lab is working on a method to induce stem cells to differentiate into insulin-producing cells. They took a stepwise approach to the problem, starting with driving embryonic stem cells toward definitive endoderm, then inducing those cells to commit to becoming pancreatic, and finally, differentiating to β-cells. The process can be accomplished by first adding activin A (a protein in the TGF-β superfamily) to the medium, followed by the small molecule, retinoic acid, and finally adding some maturation factors, said Deng.

Ultimately, the cells have to work in diabetic animals. To test their function, Deng’s group used the STZ model of acute diabetes in mice; the alkylating agent, streptozotocin (STZ) is particularly toxic to β-cells. Transplanting the differentiated cells into the liver of STZ-diabetic mice led to an increase in their survival from 20% to 80% and after two weeks the mice showed decreases in their blood sugar levels.

iPS cells can differentiate into β-cells

Since embryonic cells come from a foreign donor, graft rejection without the use of immunosuppressive drugs remains a problem. The ability to reprogram somatic cells from the patient would be one way to prevent a deleterious immune response. In 2006, Shinya Yamanaka’s group developed the iPS technique, which requires introducing the c-myc, Oct4, Sox2, and Klf4 genes into murine somatic cells to reprogram them into pluripotent stem cells. Deng’s team quickly adopted the approach but ran into difficulty when they unknowingly used an Oct4 gene with a point mutation.

Thinking that the iPS technique in humans might differ from that in mice, they looked for an additional factor that would allow them to use the procedure. They found a gene called UTF1 that is a marker of pluripotency. They also discovered that knocking down the tumor suppressor gene p53 was very helpful in increasing the efficiency of iPS cell generation. Together these two changes can replace c-myc from Yamanaka’s original four-factor approach, indicating that research into additional pluripotency factors might be fruitful. Ultimately, they were able to achieve their original goal of generating β-cells from human iPS cells, and with new innovations, they were able to get 25% of cells producing insulin, Deng said. When purified, Deng thinks these cells will produce about as much insulin as normal islet cells. However, to keep any transplanted cells healthy for the long term, the autoimmune response of type 1 diabetics will need to be suppressed.

Adult stem cells

Robert Chunhua Zhao of the Chinese Academy of Medical Sciences is investigating a particular population of pluripotent stem cells originally found in fetal tissue, characterized by the expression of the Flk-1+CD31-CD34- marker. Zhao’s group hypothesized that Flk-1+CD31-CD34- stem cell are subpopulation of pluripotent stem cells that are left over after embryonic development and have the capacity of differentiating into other types of tissue. These cells can be found in adult bone marrow and in fetal heart, lung, liver, and other tissues. Experiments in mice showed that the stem cells can differentiate into epithelial and endothelial cells as well as repopulate the lung after irradiation.

Interestingly these cells seem to have an immunomodulatory role, suppressing primary and ongoing mixed lymphocyte reactions and inducing stable mixed chimerism and donor-specific graft tolerance. To determine how the stem cells might modulate the immune response, Zhao’s group looked at the effects of these cells on the proliferation, maturation, and activation of dendritic cells, the immune cells that present antigens to T cells, thereby initiating the immune response. They found that the Flk1+ stem cells had inhibitory effects at multiple steps in the pathway toward active dendritic cells.

Because Flk1+ multipotent stem cells have an immunomodulatory effect, Zhao and his team investigated whether they could lessen the effects of graft-versus-host disease, which occurs following bone marrow transplantation with a haploidentical donor. After positive results in a primate study, they came up with a stringent clinical protocol for trials in humans that received official approval from the Chinese State Food and Drug Administration (SFDA) for Phase I (2004) and Phase II (2006) clinical trials. In leukemia patients receiving haploidentical bone marrow transplantation after their nonmyeloablative conditioning and graft-versus-host disease prophylaxis, the addition of a healthy donor's Flk1+ cells to the treatment reduced the overall incidence of acute graft versus host disease from 69.2% to 48%, and the severity was also reduced.

Incidence of any grade acute GVHD was 48% in Group A patients who had received FLK1+ stem cells in addition to standard treatment and 69.2% in Group B patients who received just the standard treatment.

The group has returned to the lab to determine the molecular mechanism of Flk1+ differentiation into hematopoietic cells, and is continuing to characterize the molecule and investigate the immune effects of these multipotent stem cells.

Driven by a desperate need for alternatives to organ transplantation and more effective treatments for a variety of diseases, research into all aspects of regenerative medicine has exploded in the past decade.

Research into all aspects of regenerative medicine has exploded in the past decade.

Pluripotent stem cells in particular have captured the public’s attention because of their potential as a new approach for treating everything from spinal cord injury to cardiovascular disease. If the development of these cells can be controlled, they could be used to replace diseased or damaged cells and tissues. Although ethical concerns have hampered research in many countries, alternatives to work with embryos have become available in the form of multipotent adult cells and induced pluripotent stem (iPS) cells derived from somatic tissue.

Several barriers to stem cell treatment remain. One problem is inherent in the very nature of stem cells—their ability to divide continuously without committing to become a specific type of mature cell means that, unless carefully controlled, they can be tumorigenic. Other major hurdles include challenges of getting stem cells to differentiate into desired cell types, organizing them into correct 3-D structures, and making essential connections with neurons and the vascular system. Further complicating matters is the fact that many organs are composed of a multitude of cell types.

Beyond stem cells

Regenerative medicine encompasses more than stem cell research. Tissue engineers, for example, are building new tissues composed of a variety of cell types in vitro using synthetic biodegradable scaffolds. To get better results, the mechanical forces that shape an organ or tissue in vivo are also being taken into account and replicated as best as possible. Meanwhile, nanoscientists are devising materials for regenerative medicine that have the right balance between biocompatibility and physical properties such as strength and flexibility.

Another area of research is xenotransplantation, in which cells or organs are transplanted from one species into another. Some believe that pigs may one day be a cheap and plentiful source of "replacement parts" for humans. However, current barriers to this approach include the possibility of cross-species infectious disease transfer, biochemical incompatibility, and immune rejection.

As tissue engineers, stem cell biologists, and others endeavor to revive or replace damaged organs, immunologists have been trying to coax the body into accepting foreign tissue. Both transplanted organs and bone marrow stem cells derived from a foreign source will elicit a potentially devastating immune response in the host. Immunosuppressive drugs are usually administered to prevent this response but the drugs leave patients susceptible to infectious disease and may fail over time. Immunologists are investigating the body’s natural immune tolerance to "self" antigens to determine how to induce tolerance to transplants. Several promising strategies have been identified.

To promote discussion about these matters and more, the New York Academy of Sciences and the Chinese Academy of Medical Sciences (CAMS) hosted a conference on regenerative medicine in Beijing, China on May 15-16, 2009. Opening the meeting, Chen Zhu, Minister of Health of the People's Republic noted, "China has attached great importance to regenerative medicine and stem cell research…and substantially increased funding in this promising area over the last few years." Researchers from Germany, Hungary, Singapore, and other countries also shared their work, highlighting the truly global nature of regenerative medicine research. In addition to reports on scientific findings, a panel of major financiers of such research in regenerative medicine discussed ways to ensure that the work is adequately funded. Another panel also offered advice to Chinese students about career opportunities in research and supporting areas.

Although it will likely require years of additional research before regenerative medicine in humans becomes routine, there is no doubt that real breakthroughs have been achieved. This conference provided a tantalizing view of the way ahead.

Speakers:
James Yoo, Wake Forest Institute for Regenerative Medicine
Yilin Cao, Plastic Surgery Hospital, Chinese Academy of Sciences
Wei Liu, Shanghai Jiao Tong University
Haiyan Xu, Center of Nanomaterials, Chinese Academy of Sciences
Ning Gu, Southeast University, School of Biological Sciences and Medical Engineering
Wei Wang, Central-South University, Cell Transplantation and Gene Therapy Institute
You-nan Cheng, Sichuan University

Highlights

• Functional bladders can be engineered using cells from the patient.
• Oxygen-releasing particles may be able to nourish cells until blood vessels reach an engineered tissue.
• Bone marrow stem cells can be induced to differentiate into chondrocytes by coculturing them with the latter cell type.
• Dermal fibroblast cells can be used to engineer tendons in vitro.
• Dynamic, rather than static, application of mechanical force to an engineered tendon is required to strengthen it.
• The composition of the scaffold is important for the development of tendons that can bear mechanical stress.
• Carbon nanotubes can increase the biocompatibility of polyurethane for use in tissue engineering.
• Porcine islet cells may be able to function in humans in the future.
• Additional characterization of porcine tissues and organs is necessary to determine their compatibility with humans.

From cells to new organ

Many researchers are trying to understand the molecular and cellular characteristics that define stem cells and their specific differentiation processes. Currently, cell therapies consist of the injection of cells at the site of tissue damage in the hope that the transplanted cells will somehow be properly incorporated and function normally. But tissues and organs are complex, heterogeneous 3-D structures. Tissue engineers endeavor to build environments that mimic the natural shape and organization of the original organ or tissue. A combination of synthetic and cellular materials may be used, and the mechanical forces that normally impact the development of the structure are also considered.

Describing his team's work on bladder reconstruction, James Yoo of the Wake Forest Institute for Regenerative Medicine presented evidence that tissue regeneration can work in humans. Yoo explained that the bladder is made up of two types of tissue: epithelial (urothelial) cells line the inside of the bladder to form a barrier that keeps urine from entering the blood stream, and muscle cells enable the bladder to contract and release the urine.

Patients have received engineered bladders constructed from their own cells.

Some people have congenital bladder defects in which the bladder is too small or their bladder pressure too high. To build a new bladder for such a patient, Yoo’s team took a small bladder biopsy, separated the two cell types, and grew large numbers of each in culture. 3D-CT images of the patient’s bladder and pelvic area were used design the bladder scaffold. Once a bladder-shaped scaffold was constructed, the researchers seeded it with epithelial cells inside the mold and muscle cells outside. Omental tissue was placed over the organ to provide vascularization. The technique was shown to be safe and effective in seven patients who received the engineered bladders. A tissue-engineering company, Tengion, is currently trying further develop this technology for commercialization.

Yoo pointed out that there are still a number of obstacles to be surmounted in the tissue engineering field. The bladder consists of only two major cell types. In contrast, the kidney is composed of over 30 different cell types, so manually placing each cell type in a specific location is not an option. Yoo described a 3D cell-printing technology in which cells are passed through the print head of an ink jet printer. By adding an "elevator" to the machine, the printer can deposit cells at a specific location along all three axes.

Two other issues that loom large are vascularization and innervation. New blood vessels generally grow at a rate of only 1mm per day. While supplying growth factors or endothelial cells that promote vascularization is helpful, it will not get around the basic growth rate problem. Yoo’s team is trying a biomaterials approach: supplying oxygen-generating particles that would allow the tissue to survive until the body’s vascular system can catch up. These particles have been shown to vastly decrease necrosis in the classic ischemic skin model and also appear to function in 3D scaffolds and gels. Yoo noted that while there is no definitive substitute for neurons yet, coculturing denervated muscle with neuronal cells appears to provide the necessary cues to keep the muscle from atrophying.

Tendon and cartilage engineering

Tendons, ligaments, and cartilage are important structural elements that have limited abilities to self-repair. These tissues may be malformed from birth, damaged by injury, or degraded by inflammatory disease. Yilin Cao and Wei Liu of the Chinese Academy of Sciences and Shanghai Jiao Tong University, respectively, are pioneers in engineering these tissues.

Vascularization is an important component of tissue engineering.

Cao has developed a method to engineer cartilage that may eventually be used to replace trachea and rebuild deformed structures such as ears. Early experiments to replace rabbit trachea revealed the importance of vascularization in the repair process. Cao’s team had hoped that they could use chondrocytes wrapped around a scaffold to form the outer cartilageneous structure and then let endogenous epithelial cells migrate inwards to form the lining. However, their engineered trachea was blocked by granulation, a type of scarring, as the wound healing process worked faster than epithelial cell growth. When the experiment was repeated with an additional step—allowing vascularization by implanting the engineered cartilage under the sternohyoid muscle—the graft performed much better.

Cao’s team also ran into difficulty when they attempted to scale up the trachea engineering procedure for larger animals. When chondrocytes are grown in tissue culture over many cell divisions, they differentiate into fibroblasts, Cao explained. So the team turned to bone marrow stem cells (BMSCs) as a cell source. The factors needed to induce BMSCs to differentiate into chondrocytes had not been identified, so Cao’s group cocultured the stem cells with chondrocytes, a process that resulted in proper differentiation of the former. Culturing them with a filter separating the two cell types indicated that soluble factors released into the culture had driven this process.

Tendon engineering

Like cartilage, tendons have high levels of collagen, but they derive from a different cell source, the tenocyte. Wei Liu described early experiments by Yilin Cao to determine whether tendons could be engineered in vitro. Cao and his colleagues began by harvesting tendon from a hen, isolating tenocytes, and expanding them in culture. They placed the cells on a biodegradable polyglycolic acid (PGA) scaffold to form a tendon-shaped structure and wrapped it with intestinal submucosa. Over time, the PGA degraded, leaving a tendon-like structure that is incorporated into the body. Cao’s team found that the engineered tendons resembled natural tendons not only in gross appearance and histologic structure but also in biomechanical properties.

Fibroblasts are able to form tendons comparable to those formed by tenocytes.

While this pioneering experiment proved that the procedure was feasible, it was clear that the researchers would need to find a more readily accessible cell type. Cao and Liu turned to dermal fibroblasts as a potential source because these cells have a similar mesodermal origin, morphology, and function. Molecular characterization showed that the extracellular matrix (ECM), growth factor expression, and collagen levels are similar between the two cell types. In a pig model, they harvested both dermal fibroblasts and tenocytes to compare the tendon-forming capabilities of the fibroblasts with that of the natural precursor. They found that the fibroblasts were able to form tendons comparable to those formed by tenocytes.

Instead of placing a tendon-scaffold construct inside the body and having the development process occur slowly over time, it would be preferable to have a mature, strength-bearing tendon available when a patient presents with an injury. Liu and Cao found that dynamic loading, in which a machine is used to bend and unbend the engineered tissue over many cycles, is critical to the proper formation of engineered tendon. However, this technique alone is insufficient to get the tendons strong enough to be functional. They turned to polymer engineers to develop composite fibers that could be used to enhance the strength of the scaffold. A ratio of 2:1 PGA to PLA turned out to have the right combination of strength and biodegradability to form good quality tissue.

Nanomaterials

Haiyan Xu at the Center for Nanomaterials at CAMS is working to develop other biocompatible materials that can be combined with cells to repair tissues. One material with useful properties is polyurethane, which is elastic and has high tensile strength. However, polyurethane doesn’t have very good biocompatibility. By adding carbon nanotubes to the material, Xu found that favorable properties can be maintained while biocompatibility is improved.

Xu is now exploring the possibility of using the composite material to mimic the basement membrane in vascular grafts. She characterized endothelial cell behavior on the nanofibrous film in terms of its secretion of basement membrane components and the activation of signal transduction pathways that respond to signaling molecules in the extracellular matrix, with promising results.

Magnetite nanoparticles form fibrous aggregates when exposed to a magnetic field.

Nanotechnology will play an important role in monitoring organ function as scientists develop new methods to repair or replace damaged body parts. Ning Gu of Southeast University, School of Biological Sciences and Medical Engineering, described the development of a number of nanoparticles and nanofibers with unique properties, such as magnetite (Fe₃O₄) nanoparticles. He has investigated the assembly of these nanoparticles using varying magnetic fields and found that a time-varied magnetic field is capable of inducing the nanoparticles into fibrous aggregates. This technique should be useful in both basic and applied research, Gu said. The nanoparticles can also be incorporated into microbubbles along with N₂ gas for use in ultrasound and as contrast agents in MRI imaging, greatly enhancing their resolution.

Xenotransplantation

Xenotransplantation offers the promise of a readily available donor source of fully developed and functional cells, tissues, and organs. Pigs are considered a good potential donor source for xenotransplantation because their biochemical profile is similar to humans. Some studies have shown that porcine insulin-producing islet cells can be used to treat diabetes in mammals, but the number of cells that must be transferred is quite large. If the islets are transplanted to the liver, the recipient risks massive bleeding, thrombosis, liver failure, and portal vein hypertension. Wei Wang of Central-South University is researching the best way to transplant islets to avoid these dangers.

He compared the transplantation of microbeads containing porcine islet cells into the portal vein versus the hepatic artery in dogs. He found that the group that received transplantation in the portal experienced necrosis and bleeding in the liver if more than 32000 microbeads per kilogram were used. The liver was almost normal in the group receiving transplantation in the hepatic artery under the same conditions. Wang’s group moved on to using islet transplantation to treat STZ-induced diabetes in rhesus monkeys. There were high levels of mortality in the portal vein group with five out of eight animals dying before the experiment was completed. Of the five monkeys that received cells via the hepatic artery and survived, four were insulin-independent up to 140 days after the transplant. Although success can be achieved with the procedure, Wang indicated, more work needs to be done in model animals before moving into humans.

Most porcine cells express antigens that will be recognized by the human immune system as a threat to the body. You-nan Cheng of Sichuan University is working to characterize the antigens that stimulate the hyperacute rejection of porcine tissue in primates. The main focus of research had been the α-Gal antigen, which is present on the surface of endothelial and epithelial cells in pigs. After generating transgenic pigs that should lack the antigen, they found that the tissue was still rejected, suggesting that additional antigens are contributing to the graft rejection and/or that the gene that was knocked out was not entirely responsible for the generation of the antigen. Studies are ongoing to explore both possibilities.

Assuming that the immune system doesn't reject a xenograft, it is still necessary to determine if the foreign organ functions similarly enough to the endogenous organ to take its place. Cheng is comparing the biochemical function of porcine and human livers to determine if the porcine organ could be used as a replacement.

Speakers:
Ibrahim Domian, Massachusetts General Hospital
Ulrich Martin, Hannover Medical School

Highlights

• Induced pluripotent stem cells (iPS cells) can differentiate into cardiomyocytes.
• Bone marrow mononuclear cells can improve survival outcomes in acute myocardial infarction patients.
• Mononuclear cells contribute to the vascular lineage when injected into damaged murine hearts.

The right source

Cardiovascular disease (CVD) is a growing problem worldwide. While already a major killer in wealthy nations struggling with obesity, diabetes, and high blood pressure, the World Health Organization estimates that by 2010, CVD will be the leading cause of death in developing countries too. Once prevention methods such as diet and exercise fail, treatment of heart disease is costly, invasive, and eventually unsuccessful. Regenerative medicine targeting the heart and vasculature is on everyone’s wish list.

Stem cell therapies to repair the heart have already been tried, but they have not been very successful. Although pluripotent stem cells should, by definition, be able to reconstitute any organ, researchers do not yet have a thorough enough understanding of organ and tissue development to determine what cues need to be provided to make this occur.

Ibrahim Domian of the Richard B. Simches Research Center at Massachusetts General Hospital is filling these gaps in the understanding of heart development. He and his colleagues have devised a system to label cardiac progenitor cells and follow their progress in living animals. The details of his approach will be described in a forthcoming publication.

iPS cells as a source of cardiomyocytes

Producing cells in sufficient quantity for use in regenerative medicine remains one of the field’s greatest challenges. Use of embryonic stem cells is hampered by ethical concerns and adult stem cells remain of questionable value. Complicating this issue is the fact that cells that are foreign to the body in which they are implanted may be rejected by the recipient’s immune system.

In 2006, Shinya Yamanaka’s group developed a new kind of stem cell that gets around all these issues, called induced pluripotent stem cells (iPSCs). They identified four transcription factors that were active in embryonic stem cell lines and transfected the genes into mouse fibroblast cells using a retroviral vector. The method was extremely successful in reprogramming the cells into stem cells, though it posed a few problems that are slowly being overcome.

One problem with the approach is that the adult somatic cells used as a source accumulate mutations over time that persist in the iPSCs. For this reason, Ulrich Martin and his team at Hannover Medical School turned to umbilical cord blood, specifically endothelial cells, as their starting material for regenerating heart muscle. Their iPSCs showed a stable karyotype over time and high expression of endogenous pluripotency factors, both important for ensuring that cell quality doesn’t deteriorate over time.

iPS cells can differentiate into cardiomyocytes. Now the challenge is to grow them in large numbers.

The team has investigated whether, like embryonic stem cells, cord blood iPS cells are capable of differentiating into cardiomyocytes. To get stem cells or iPS cells to differentiate, researchers grow the cells in suspension in the absence of antidifferentiation agents, using the “hanging droplet” method. When cultured in this way, the cells aggregate and spontaneously differentiate into structures called embryoid bodies. Embryoid bodies recapitulate many of the features of the developing embryo, including differentiation into all three germ layers (endoderm, mesoderm, and ectoderm). Among the cell types that develop in the embryoid bodies are cardiomyocytes that beat rhythmically.

To ensure that the iPSCs could be a reliable source of cardiomyocytes, Martin’s team thoroughly characterized the differentiation process. They observed that the expression level of the pluripotency factors dropped as the cells differentiated, as expected, although they saw some transgene expression in some of the clones. Markers for the different germ layers showed that the iPSCs were capable of forming all three layers. The cardiomyocytes that developed in the embryoid bodies had the calcium transient coupling, β-adrenergic receptors, and field potentials characteristic of normal cardiomyocytes. The group is now trying to scale up the cultures for both pharmacological screening and potential clinical applications.