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Biologic and Materials Sciences and Division of Prosthodontics

Krebsbach Lab Current Research

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I. Functional Characterization of the Stem Cell Niche (NIH R01 DK082481)

Although a population of bone marrow stromal cells (BMSCs) has been referred to as mesenchymal stem cells (MSCs), it is clear that this population is heterogeneous and likely contains several cell types including few, poorly discernible stem cells. However, our group is now able to prospectively identify FACs sorted MSCs and demonstrate their stem cell activity in vivo prior to any cell culture in vitro. Our experiments are designed to understand the nature of these MSCs and how their microenvironment, including interactions with hematopoietic stem cells (HSCs), influences their biologic activity. The overall hypothesis is that we have identified a true mesenchymal stem cell within the bone marrow and the activity of this stem cell - including interactions with hematopoietic stem cells - is dependent on its niche compartment.

We have identified a partially purified mesenchymal stem cell (MSC) population that maintains its multi-lineage potential both in vivo and in vitro.  Most importantly, we demonstrated that as few as 500 purified cells can develop bone in vivo without prior cell expansion in culture. This prospective isolation process sets us apart from others in the mesenchymal biology field and will allow us to understand the crucial differences in stem cell properties between cell culture and in vivo environments. In this project we will study the biology and in vivo function of this population and compare its activity to the frequently studied and more heterogeneous bone marrow stromal cell population.

The overall hypothesis behind our proposed projects is that a true mesenchymal stem cell exists within the bone marrow and the activity of this stem cell - including interactions with hematopoietic stem cells - is dependent on its niche compartment.

To this end, we propose the three aims listed below. Each aim offers the opportunity to generate new tools that will revolutionize the ability to discover and model the environmental factors that mediate cell function in complex in vivo systems such as the bone/bone marrow.

Aim 1.  To identify and characterize cells within the bone marrow stroma that exhibit mesenchymal stem cell activity. We intend to answer the following compelling questions: 1) Do these purified cells function as stem cells in vivo? 2) What is the capacity of these purified cells to function in their native in vivo micro-environment? and 3) How do MSCs respond to physiologic stimuli?

Aim 2.  To identify the MSC niche and determine if the MSC pool and its niche change under physiologic conditions  (Mapping and defining the niche). We will address the following experimental questions 1) Where in the marrow does the MSC reside? 2) Does MSC numbers change in response to physiologic and or pathologic stimuli including a single dose of 5-FU, a single acute bleed and anabolic PTH treatments. 3) Does perturbations of the marrow alters/regulates the MSC phenotype. 

Aim 3.  To determine the functional relationship between HSCs and MSCs  (Studying the cross – talk between HSCs and MSCs). 1) Do MSCs and HSCs co-localize to the same niche? 2) Do HSCs (isolated via the SLAM family markers) regulate MSC (very small, Sca-1+lin-CD45-) fate using the most highly purified cells available. 

II. Engineering Multi-Tissue Interfaces (NIH R01 DE018890)

When congenital anomalies, traumatic injuries or inflammatory and degenerative diseases involve an articulating joint such as the temporomandibular joint (TMJ), the effects are often physically, financially and emotionally debilitating.  Unfortunately, despite decades of targeted clinical and basic science research, well established methods to repair or regenerate such joints remain elusive, resulting in a significant unmet clinical need. Although the last two decades have seen a greater emphasis on the scientific underpinnings of tissue engineering, current reductionist approaches give a fragmented view of the future by investigating the regeneration of single tissues or materials.  This limitation is troublesome because cells and tissues do not function independently in the context of living organisms.  To address this problem, we have aligned a multidisciplinary team to investigate one of the next frontiers in tissue engineering, that is, the development of multi-tissue interfaces.

The long-term objective of this proposal is to develop strategies to regenerate multi-tissue interfaces with a focus on the bone-cartilage interface.  While our experimental models will concentrate on the osteo-chondral interface found in articulating joints, the principles developed here may subsequently be applied to the regeneration of many other tissue interfaces.  We propose a hypothesis and design-driven, integrative tissue engineering project based on rapid fabrication of bioengineered scaffolds, with custom-tailored surface chemistry, that control the spatial and temporal release of bioactive factors to regenerate the bone and cartilage interface.  The controlled generation of this interface will be directed via an in vivo regenerative gene therapy approach.  The central hypothesis is that delivery of bioactive signaling factors (BMP-2 and Sox9) to distinct regions of designed scaffolds can control the lineage commitment of responsive cells to develop a bone/cartilage interface.

Aim 1.    To custom-tailor the surface chemistry of biomaterials with precisely designed biological signaling properties.

Hypothesis: Viruses immobilized by bioconjugation on scaffold surfaces can spatially control cell transduction in discrete regions of designed biomaterial scaffolds.

Experimental approach:  Poly ε-caprolactone (PCL) surfaces will be modified by chemical vapor deposition (CVD) to establish surface coatings with a variety of polymer properties and conjugation chemistries.  We will prepare polymer carriers to control binding of two or more cell-signaling factors in defined loading ratios.  For this reason, co-polymers will be developed and protocols for bioconjugation of two or more different adenoviruses will be tailored to achieve cell transduction directly from the biomaterial surface. Three different immobilization models will be developed in this specific aim to gain maximal control of viral release through a dynamic equilibrium of biotin/avidin and biomaterial interactions.

Aim 2. To immobilize two different viruses on a single scaffold to control delivery of specific biological signaling factors and to understand how these signals control the development of a biological interface.

Hypothesis:  Modifying the surface chemistry of designed biomaterials will allow multiple viral vectors encoding biological signals to direct and precisely control a biological interface.

Experimental approach: Material surfaces will be modified to control the delivery of multiple adenoviruses.  Discrete regions of functionalized scaffolds will be physically or photochemically masked to create defined regions for virus immobilization and the generation of a cell-signaling interface.  Two-way CVD will also be used to generate signaling gradients to mimic natural developmental signaling patterns at an interface.

Aim 3. To develop a bone-cartilage interface by directing the lineage progression of responsive cells to bone and cartilage using in vivo regenerative gene transfer strategies on designed biomaterial scaffolds.

Hypothesis: The delivery of bioactive signaling factors to distinct regions of a designed scaffold can control the lineage commitment of responsive cells to either bone or cartilage and generate a functional tissue interface in 3-dimentional scaffolds.

Experimental approach:  Tissue interfaces will be generated on biomaterial scaffolds in vivo. The precision of interface development will be studied by delivering BMP-2 on one region of a scaffold and antagonists such as noggin or dominant negative BMP receptors on the adjacent surfaces. The development of bone/cartilage interfaces will be studied in vivo by the controlled delivery of BMP-2 (bone) and Sox-9 (cartilage).

III.  Osteoblast Lineage Progression from Human Embryonic Stem Cells (NIH R01 DE  016530)

Current practices to maintain induced pluripotent stem cells (iPS) and human embryonic stem cells (hES) in an undifferentiated state typically require the support of mouse embryonic feeder cells (MEFs) or an undefined extracellular matrix such as Matrigel™. These culture conditions that depend on animal contaminants, severely limit our ability to interpret mechanistic studies designed to resolve how human pluripotent stem cells interact with their extracellular environment to: 1) remain in a unique undifferentiated state and 2) make fate-changing lineage decisions. Likewise, the xenogeneic components of MEFs and Matrigel™will ultimately hinder our ability to use these stem cells to treat debilitating human diseases.

We have overcome these obstacles by developing a synthetic matrix system that supports iPS (and hES) cell expansion and self renewal within completely defined culture conditions that are free from xenogeneic contamination(Nature Biotechnology (1), Nature Protocols (2)).The establishment of this synthetic matrix now allows us to probe the molecular basis of pluripotent stem cell self-renewal and differentiation, and will pave the way for clinical applications. Compared to genetically and immunologically incompatible animal-derived matrices, this polymer (PMEDSAH, “poly-med-sah”), has the advantages of being: 1) synthetic, 2) defined, 3) reproducible, 4) capable ofsynthetic modification to take on new properties, 5) resistant to degradation, and 6) unlike recombinant protein-based matrices, can be subjected to long-term storage and sterilization.

Most research groups use biological components such as RGD peptides or natural sugars to mediate the interaction between cells and substrates. Our innovation is the development of synthetic components that function as the primary structural motifs in stem cell-to-substrate interactions. This biomimetic approach provides a material that has both physical and chemical properties that can be readily modified to permit us to understand, and perhaps control, how synthetic extracellular biomimetics modulate important biologic activities such as self-renewal and pluripotency. This cannot be accomplished using currently available approaches and our interdisciplinary group is uniquely positioned to take on this important scientific problem (Dr. Lahann - Chemical Engineering, and Dr. Krebsbach - Stem Cell Biology).  Our long-term goal is to define the molecular mechanisms that allow iPS cell self-renewal on PMEDSAH and the controlled differentiation of these cells towards a mesenchymal stem cell (MSC) phenotype. Accomplishing these goals is an important prerequisite to the development of therapeutic protocols using pluripotent stem cells to regenerate human tissues. Our goals and hypotheses will be tested by performing experiments described in the following Aims.

Aim 1.   Define the structural and/or physico-chemical properties of PMEDSAH that lead to iPS cell self-renewal and maintenance of the undifferentiated state

Hypothesis:  The physical and chemical properties of PMEDSAH facilitate iPS cell self-renewal and maintenance of the undifferentiated state.

Experimental Approach: Structure/function analysis will be performed with the highly manipulatable PMEDSAH substrate and pluripotent iPS cells using the following experimental perturbations: 1) modify material stiffness, charge density and contact angle by controlling polymer thickness, 2) modify polymer structure and chemistry (linkage between polymer backbone and side chain, distance and molecular position of the zwitterionic groups) and the type of anionic end groups (sulfonate vs. phosphonate groups). iPS cell adhesion, proliferation and self-renewal will be assayed on the modified surfaces.

Aim 2.   Determine the cell receptor mechanisms that direct adhesion and maintain iPS cells in an undifferentiated state on synthetic polymer substrates

Hypothesis:  Pluripotent iPS cells use more than one cell adhesion system to adhere to and support self-renewal on a defined, synthetic polymer substrate.

Experimental Approach: While Aim #1 focuses on the physico-chemical properties of the substrate, Aim #2 takes the converse approach and is designed to define the properties of the cell surface as iPS cells interact with PMEDSAH. Our objective is to use our novel synthetic polymers as a tool to understand the molecular basis of adhesion and self-renewal of iPS cells using three experimental strategies: 1) cell adhesion to solid phase substrates via integrins, 2) an alternative mechanism of cell adhesion using heparin/heparan sulfate (HS) and heparin sulfate proteoglycan (HSPG) binding, and 3) a proteomics approach using metabolically-labeled subcellular fractions to determine the extent to which other cell adhesion systems are used to support self-renewal of iPS cells on PMEDSAH.

Aim 3.   Determine the extent to which iPS cells maintained on PMEDSAH are capable of subsequent lineage-specific differentiation and regeneration of clinically relevant craniofacial skeletal defects

Hypothesis:  Patient-specific iPS cells can be derived on fully defined and xeno-free PMEDSAH and be reproducibly differentiated into mesenchymal stem cells (MSCs) to regenerate skeletal defects.

Experimental Approach:  Patient-specific iPS cells will be generated from somatic cells. We will 1) generate three patient-specific iPS cell lines on PMEDSAH, 2) direct iPS cell differentiation to MSCs in vitro while also demonstrating the loss of iPS cell phenotype and function, and 3) determine the extent to which iPS-derived MSCs regenerate bone in clinically relevant craniofacial skeletal defects.

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