Identification of Molecular and Cellular Pathways Involved in Differentiation of Stem Cells into Functional Tissues Shahin Rafii's laboratory focuses on stem cell biology and angiogenesis uses in vivo mouse model and mouse and human genetics, tissue culture approaches and molecular biology to model angiogenesis, cancer and stem cell metabolic regulation. Genetic, genomic, molecular and cell biological techniques are combined to achieve a systems level understanding of these complex processes.
Currently, Rafii's work is focused on identifying the molecular and cellular pathways involved in organ regeneration and tumor growth. He has established the concept that vascular endothelial cells are not just inert plumbing to deliver oxygen and nutrients, but also by production of tissue-specific growth factors, defined as angiocrine factors, support organ regeneration and tumor proliferation. He has shown that bone marrow endothelial cells by elaboration of angiocrine factors, such as Notch ligands, support stem cell self-renewal and differentiation into lymphoid and myeloid progenitors. He has recently demonstrated that liver and lung endothelial cells are endowed with unique phenotypic and functional attributes and by production of unique instructive growth factors contribute to the hepatic and alveolar regeneration.
He employed this knowledge to induce differentiation of the murine and human pluripotent embryonic stem cells into functional and engraftable vascular and hematopoietic derivatives. He developed screening approaches to exploit endothelial cells as a vascular niche platform to identify as yet unrecognized novel angiocrine factors that instruct tumor proliferation as well as orchestrating stem cell self-renewal and differentiation. As such, studies of c-ets-1, Fos, Foxo and Nrf2 dependent signaling and gene regulation in oxidative stress response and stem cell regulation on signaling pathways that control differentiation and de-differentiation of stem cells in vitro and in vivo models, and the role of matrix metallo proteases in developmentally controlled lung generation as well as pathological cell movements and tissue invasiveness. He is developing pre-clinical and clinical models to interrogate the potential of organ-specific endothelial cells in repairing injured organs or to target tumor vasculature.
studies of stem cells
Stem cells derived from adult tissues are endowed with the unique capacity to regenerate functional organs. Multipotent stem cells not only undergo self-renewal but also differentiate under permissive conditions into lineage-specific progenitors that could potentially revitalize every organ. Adult organs-including bone marrow, myocardium, vascular system, liver, and lung-harbor renewal sources of endogenous stem cells that could contribute to the regeneration of damaged tissue. Adult bone marrow is also a rich reservoir of hematopoietic and vascular stem and progenitor cells.
As the stem cell reservoir in the adult organs is scant, it may be difficult, using current technology, to obtain sufficient numbers of transplantable stem cells that could be used clinically. Alternatively, embryonic and fetal stem cells, as well as reprogrammable germline stem cells, are a rich source of stem cells that can be used for therapeutic organ regeneration and revascularization. Furthermore, because stem cells can support tumor growth and metastasis, understanding how tumor tissue recruits tumor stem cells and vascular progenitors will make it possible to design new strategies to block tumor growth and inhibit tumor neo-angiogenesis.
Despite the tremendous potential for the use of adult or embryonic stem cells for organ regeneration or to target tumor tissue, the molecular and cellular pathways that support recruitment and differentiation of stem cells into functional organs remain unknown. Furthermore, recent setbacks with stem cell therapy for myocardial regeneration suggest that breakthroughs are necessary to take advantage of stem cells and their progeny for organ regeneration and repair.
Significant hurdles must be overcome, however, to facilitate the introduction of stem cells into the clinic for organ regeneration and revascularization: Adult stem cells are scarce and thus their use for generation of large tissues is not practical. Adult stem cells are primarily programmed to generate a very specific set of tissues, and thus have limited multipotentiality. Culture conditions that will promote differentiation of stem cells into functional tissues must be identified.
Rafii's lab has developed a number of strategies to circumvent these obstacles.
new sources of stem cells
Despite initial enthusiasm to use adult bone marrow stem cells for organ regeneration, evidence suggests that adult bone marrow contains limited numbers of organ-specific stem cells. Adult bone marrow has sufficient numbers of stem cells to reconstitute hematopoietic cells but diminishing numbers of stem cells to contribute to vascular regeneration. Other sources of organ-specific stem cells to generate vascularized pancreatic islet cells, myogenic cells, or neuronal cells may be necessary to treat diabetes, myocardial infarction, or stroke. Human embryonic stem cells provide a rich source of stem cells for therapeutic purposes. In collaboration with Zev Rosenwaks (Cornell University Weill Medical College), we have generated and cultured two validated human embryonic stem cell lines-Weill-Cornell-1 (WC-1) and WC-2. We are establishing protocols to generate functional blood vessels from human embryonic stem cells.
In a remarkable breakthrough, we have identified another source of stem cells that may be used therapeutically for organ regeneration. We have isolated large numbers of mouse spermatogonial stem cells and differentiated them into adult multipotent cells, with the potential to differentiate into endothelial cells, myogenic cells, and neuronal cells. We are examining the potential of spermatogonial stem cells derived from humans to differentiate into autologous human adult multipotent cells.
Scalability. If the goal of stem cell therapeutics is to generate vascularized human tissues, substantial numbers of stem cells will be necessary. Therefore, stem cells with the capacity to undergo expansion are ideal sources of cells for therapeutic organ revascularization. Current hurdles in expanding human adult stem cells limit the amount of tissue that may be necessary to regenerate a damaged organ or target tumors. Therefore, human embryonic stem cells, which can be expanded in large numbers, are an indispensable source of transplantable human tissue. One advantage of spermatogonial-derived cells is that they allow for generation of large numbers of autologous spermatogonial stem cells, circumventing the immune rejection or graft-versus-host responses.
Circumventing the need for a genetic match. Currently, bone marrow transplantation for hematological disorders has one major shortcoming: in many cases the proper genetic match is not available. Human embryonic stem cells, especially spermatogonial stem cells, allow for generating autologous stem cells for therapeutic organ regeneration. Development of human embryonic stem cell or spermatogonial stem cell technology will have a tremendous impact on the treatment of a wide variety of patients.
Despite availability of sophisticated bone marrow registry and blood banks, there are many patients with leukemias, lymphomas, or solid tumors who succumb to their disease because a genetically suitable match is not available. Many patients also receive marrow that is not a perfect genetic match, resulting in disabling graft-versus-host disease.
Derivation of human embryonic, spermatogonial, and oogonial stem cells could provide an unlimited source of genetically matched autologous stem cells for organ regeneration and tumor targeting, as well as treatment of genetic disorders, including diabetes, heart disease, stroke, and vascular diseases. We are also investigating tumor vessel targeting, which could be applied to common cancers, including lung, breast, esophageal, and colon cancers.
Our lab is studying the potential of stem cells for therapeutic organ vascularization in three models:
1. Lung regeneration: Removal of the left lung of the mouse results in the regeneration of the remaining right lung. We have shown that marrow-derived progenitors contribute to the revascularization of the remaining right lung.
2. Liver regeneration: Partial hepatectomy promotes recruitment of liver oval stem cells expressing VEGF-R1 (vascular endothelial growth factor receptor-1) to contribute to neo-angiogenesis and liver regeneration. This model allows us to examine the role of hemangiogenic progenitors and preconditioning in organ regeneration.
3. Cardiac and ischemic limb preconditioning: Acute ischemia induced by the ligation of the left anterior descending coronary artery or femoral artery will allow us to examine the role of preconditioning of the recipient neo-angiogenic niche with PDGF (platelet-derived growth factor) and/or BDNF (brain-derived neurotrophic factor) in incorporation of the hemangiogenic progenitor cells into ischemic tissues.
de novo generation of human haematopoietic stem cells.
Development and maintenance of the haematopoietic system relies on a scant number of self-renewing haematopoietic stem cells (HSCs) residing in the adult bone marrow and representing the top of a complex cellular hierarchy. Transplantation of HSCs, harvested from either bone marrow, mobilized peripheral blood or umbilical cord blood (UCB), has become the standard of care for numerous hereditary and malignant blood diseases. However, the limited availability of optimally human leukocyte antigen (HLA)- matched donor HSCs remains a challenge, especially for individuals of non-Caucasian background or mixed ethnicity. While the immunologic naïveté of UCB enables transplantation despite antigen mismatch, the relatively low HSC dose slows engraftment and raises the threat of graft failure. In vitro expansion of UCB HSCs has been vigorously investigated, but despite substantial progress, current protocols are not yet clinically approved. Consequently, and because considerable interest in illuminating fundamental aspects of blood development, de novo generation of HSCs from non haematopoietic sources has become a major objective for the field — a ‘holy grail’ — with wide-ranging implications for HSC biology and transplantation medicine. Here we are summarizing our latest efforts and progress towards de novo generation of bona fide haematopoietic stem cell.
HSCs have been shown to arise from specialized endothelial subpopulations with hemogenic potential. In animal models, the transition from endothelial to hematopoietic identity has been directly observed both using live and explanted embryonic tissues; however, analogous studies using human embryos are handicapped by technical and ethical obstacles. Human embryonic stem cells (hESCs) provide an in vitro platform for studying the initial events involved in the differentiation of hematopoietic progenitor cells (HPCs). But a major impediment to the isolation, expansion, and study of hESC-derived HPCs is that their putative cells of origin, hemogenic endothelium, exist only ephemerally, and the early stages of hematopoietic ontogeny in this context have not been described.
A clear demarcation of the ontogeny of hematopoietic cells arising from hESCs would enable the integration of in vivo and in vitro developmental studies and accelerate efforts to generate therapeutically useful cell types. To these ends, we generated a transgenic hESC line that separately identifies emergent endothelial and hematopoietic cells during differentiation and used it to directly observe the spectrum of phenotypic progression from hemogenic endothelium to multipotent HPCs and their derivatives. Live imaging of hemogenic ECs during endothelial to hematopoietic transition (EHT) and subsequent differentiation identified phenotypic milestones during hemato-endothelial specification and revealed a temporal bias in lineage potential that correlates with discreet waves of hemogenesis noted in mouse and human fetal tissues.
Despite recapitulating key developmental branching points and acquiring phenotypical attributes of haematopoietc stem cells, our efforts did not yield engraftable HSCs1. Therefore, to generate such cells, we decided to re-ignite “hemogenic memory” in adult endothelial cells by overexpressing key transcription factors.
Direct conversion of cellular identities through transcription factor (TF)-mediated reprogramming represents an alternative strategy to directed differentiation. This approach allows the cell fate of interest to be obtained through the expression of key cell-fate determining TFs. To circumvent transition through a destabilizing pluripotency state, attempts have been made to reprogram non-haematopoietic cell types into HSCs but these efforts produced haematopoietic progenitor-like cells with poor engraftment potential.
The inability to generate HSCs could be explained by lack of proper environmental cues to self-renew reprogrammed HSCs. We successfully reprogrammed human umbilical vein endothelial cells (HUVEC) to engraftable HSC-like cells through direct conversion by constitutive expression of FOSB, GFI1, RUNX1, SPI1 (FGRS). Propagation of these cells onto a vascular-niche-like environment substantially enhanced reprogramming efficiency, emphasizing the importance of inductive cues from the physiological micro-environment in the orchestration of haematopoietic specification. The converted cells acquired colony-forming potential and were successfully engrafted in immuno- deficient mice after primary and secondary transplantation, producing long-term myeloid and B lymphoid progeny. However due to constitutive expression of SPI1 and poor recipient lymphoid competence, we could not generate and mature T cell progeny.
Using a conditional expression of FGRS along with congenic transplantation assay, we were able to prove that transient expression of these four transcription factors in adult ECs along with co-culture with proper vascular microenvironment was sufficient to generate HSCs with transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, clonal engraftment and serial primary and secondary multi-lineage reconstitution, and restoring antigen-dependent adaptive immune function3.
Conversion of adult mECs to HSCs appears to be efficient. We showed that a single clone of runx1+ FGRS-EC yield 598 ± 463 bona fide haematopoietic stem cells. Considering we are obtaining 70,000 runx1+ FGRS-EC per conversion, we could theoretically have achieved a clinically translatable dose of 24,000 bona fide HSCs.
targeting the premetastatic niche
In collaboration with David Lyden (Cornell University Weill Medical College), we have discovered that nonmalignant bone marrow cells establish "cellular bookmarks" in target organs that guide cancer cells to their predetermined destination. This finding could have a major impact on how oncologists assess the likeliness of metastasis to specific organs. This discovery may also help identify subsets of high-risk cancer patients who are prone to distant metastases. Those patients would likely benefit from a more aggressive adjuvant therapy. Understanding how cellular bookmarking works at the molecular level could lead to new information that may help thwart metastasis, a major cause of death among cancer patients.
We have established that a specific subset of bone marrow–derived cells (BMDCs)-which are composed of hematopoietic progenitor cells capable of dividing and forming colonies-are recruited by tumors to aid in the growth of new blood vessels. The generation of new blood vessels occurs through a process called angiogenesis. In previous studies, we had shown that corecruitment of hematopoietic BMDCs expressing the angiogenic factor receptor VEGF-R1, along with the vascular cells, accelerated the assembly of newly formed blood vessels and tumor growth.
We have demonstrated that a nonmalignant cluster of VEGF-R1 hematopoietic BMDCs is recruited to a premetastatic niche, thereby establishing a docking site prior to the arrival of the circulating tumor cells.
In experiments with mice that had been implanted with highly metastatic lung cancers or melanoma cells, we discovered that BMDCs do, indeed, arrive at the premetastatic sites before the arrival of cancer cells. We also found that such clusters appear prior to the development of metastases in mice genetically predisposed to developing tumors-a system that closely mimics how cancers develop.
Interference with the mobilization of VEGF-R1 cells from the bone marrow and incorporation into the premetastatic niche significantly decreased subsequent tumor metastasis. Moreover, depleting VEGF-R1 cells or inhibiting the function of VEGF-R1 itself also retarded the spread of tumors to their predestined metastatic sites. Remarkably, tumor types determined the pattern of organ localization of BMDCs. By releasing soluble factors, tumor cells were directing BMDCs to spread to the sites where they were supposed to go. For example, melanoma cells that have the capacity to metastasize to virtually every organ, released factors that directed incorporation of VEGF-R1 BMDCs to all of the organs that are known to be the common sites for melanoma metastasis.
We also identified a number of regulatory molecules, including the adhesion molecule VLA4 and the protease MMP-9, which are necessary for BMDCs to establish the premetastatic niches in target organs and for tumor cells to find and attach to those niches. VLA4 enables attachment of BMDCs to components of tumor stroma, such as fibronectin. Soluble factors released by the tumor cells selectively stimulated the production and deposition of a matrix molecule, fibronectin, which provided a docking site for the attachment of BMDCs before arrival of the tumor cells.
In another clinically relevant observation, we found numerous VEGF-R1 clusters in various tissues obtained from patients with breast, lung, and esophageal cancers. Conventional diagnostic techniques, such as light microscopy, may fail to detect very small micrometastatic tumors in the lymph nodes in the immediate vicinity of the primary tumor. But the presence of VEGF-R1 clusters might indicate undetected micrometastases, or impending metastasis, which would suggest that these particular patients may be at higher risk for tumor metastasis and thus should be treated more aggressively with adjuvant chemotherapy.
These findings also suggest that tumor metastatic potential may not only depend on the oncogenicity of the cancer cells but also on the existence of developmentally "premetastatic niches" or "hot spots" in the body that are receptive to metastatic cells.
It is conceivable that the number and capacity of these hot spots to permit attachment of tumor cells may be determined by the genetic makeup of any given patient. For example, the propensity and magnitude of incorporation of VEGF-R1 into various organs may differ from one patient to another, and might explain why subsets of patients with early-stage colon cancer are more prone to liver metastasis, while others with an identical stage of cancer and oncogenic repertoire are cured of their disease with timely surgery and adjuvant chemotherapy.
When oncologists diagnose a tumor at an early stage they face a dilemma about what to do after the surgeon has removed the tumor, particularly when pathological examination does not show any evidence of microscopic metastasis. For example, on average, only 30 percent of patients with fully resected primary tumors may relapse, while others are most likely cured of their disease. It is unnecessary to expose patients at low risk for relapse to high doses of toxic chemotherapy, which is usually associated with significant morbidity. There is a possibility, however, that the presence of VEGF-R1 hematopoietic BMDC clusters in the resected "tumor-free" tissues portends poor prognosis; these patients may benefit from treatment with aggressive chemotherapy.
regulation of hematopoiesis by microvascular endothelium
The hematopoietic microenvironment is critical for the self-renewal, proliferation, and differentiation of pluripotent hematopoietic stem cells. Within the hematopoietic microenvironment, whether it is embryonic yolk sac, fetal liver, or adult bone marrow, microvascular endothelium not only acts as a gatekeeper controlling the trafficking and homing of hematopoietic progenitors, but also provides cellular contact and secretes cytokines that allow for the preservation of the steady state hematopoiesis. We have developed a technique for the isolation and cultivation of adult bone marrow microvascular endothelium (BMEC) and fetal liver endothelial cells (FLEC). We have shown that BMEC and FLEC monolayers support the trafficking as well as long-term proliferation and terminal-differentiation of CD34 hematopoietic progenitors. Direct cellular contact between endothelial monolayers and progenitor cells enhances the survival and regeneration of pluripotent hematopoietic progenitor cells. Although we have shown that binding of CD34 progenitor cells to BMEC monolayers is partially mediated through interaction between CD34/L-selectin, b1, b2 integrins, and membrane-bound kit-ligand/c-kit receptor ligand pairs, as yet unrecognized membrane-bound adhesion molecule/chemoines are responsible for the self-renewal and homing of the pluripotent stem cells.
The major focus of our laboratory is to isolate and characterize known and novel adhesion and membrane-bound cytokines expressed by endothelium that regulate proliferation and adhesion of hematopoietic stem cells and their progenitors. To this end, we have utilized expression cloning strategy using BMEC and FLEC cDNA libraries to screen for known and novel adhesion/homing receptor and membrane-bound cytokines that regulate proliferation of hematopoietic progenitors. In collaboration with Dr. R. Crystal, adenoviral vectors overexpressing cytokines, and adhesion molecules are being used to examine their function in long-term CD34 progenitor-endothelial coculture studies. Direct introduction of adenoviral vectors expressing stem cell active cytokines, into hematopoietic microenvironment provides novel approaches for the treatment of acquired or congenital hematological disorders.
Neo-angiogenic and stromal profiling of Cancer tissue
Based on the hypothesis that autocrine and paracrine VEGF-A and VEGF-receptor signaling support proliferation and tissue invasive potential of subsets of breast cancer cells, breast cancer activation and proliferation of VEGFR2 and VEGFR3 endothelial cells results in the release of paracrine factors that supports in turn the growth of breast cancer cells. This is based on our previous demonstration that functional VEGF-receptors including VEGFR1, are expressed on subsets of breast cancer cells generating an autocrine loop that is essential for the survival, and progression of subsets of breast cancer cells. Therefore, inhibition of VEGFR/VEGF-A autocrine and paracrine pathways may be effective in inducing apoptosis of proliferating endothelial cells as well as in inhibiting the growth of breast cancer cells.Emerging evidence also suggests that VEGFR1 may also be expressed on the breast cancer SUPPORTIVE tissue. Therefore, targeting the breast cancer stromal tissues may provide a novel means to enhance anti-angiogenic and anti-tumor effect of chemotherapy.
ongoing preclinical and clinical trials
Our lab is directly involved in a number of preclinical and clinical trials.
The role of anti-angiogenic factors in the treatment of VEGF-R2 leukemias: Presently, there are several ongoing clinical trials at Weill-Cornell Medical Center and worldwide where the role of VEGF-R2 tyrosine kinase inhibitors, Avastin (anti-VEGF-A), and other anti-angiogenic agents are being studied for the treatment of refractory acute myelogenous leukemias. Our lab is validating the response to anti-angiogenic agents by studying the leukemic cells in vitro and assessing their response to VEGF-A.
Hemangiogenic stem and progenitor cells as surrogate markers for evaluating response to anti-angiogenic agents: Angiogenic factors released by the tumor tissue or ischemic organs induce mobilization of hematopoietic and vascular stem cells to the peripheral circulation. The absolute number of these cells detected during their sojourn in the peripheral circulation correlates closely with the endogenous neo-angiogenic activity. Quantification of the mobilized VEGF-R1and CD133 VEGF-R2 hemangiogenic stem and progenitor cells provides a novel means to assess the angiogenic response to anti-angiogenic agents or chemotherapeutic agents. We are investigating the role of these vascular surrogate markers in patients with colon, breast, and brain tumors.
Acceleration of wound healing by autologous bone marrow transplantation: My group is investigating the role of bone marrow–derived cells in the regulation of wound healing. In an FDA-approved protocol, the marrow of patients with a full-thickness burn (third-degree burn) is harvested and applied to the debrided wounds in conjunction with artificial skin allografts. This study tests whether marrow-derived cells can facilitate revascularization and regeneration of the damaged skin. It also provides the platform for future studies of the potential benefits of preconditioning damaged skin tissue to enhance engraftment and differentiation of marrow-derived cells.
Targeting tumor neovessels with genetically manipulated pro-angiogenic hematopoietic and endothelial progenitor cells: Based on published data and data generated in our laboratory, circulating hemangiogenic progenitors will provide an effective means to target tumor vasculature. It remains to be determined, however, whether such an approach is feasible and effective in a clinical setting. We plan to obtain the peripheral blood of patients with underlying malignancies and isolate pro-angiogenic progenitors. Re-introduction of these tumor-seeking cells armed with anti-angiogenic factors, such as thrombospondin-1, will allow selective targeting and destruction of the tumor neovessels.