Research

	Stem-Cell & Hematopoietic Tissue Engineering: Cell Biology, DNA Arrays & Models
	Metabolic Engineering, DNA Arrays and Functional Genomics of Solventogenic Clostridia
	T-Cell Based Cellular Immunotherapies & Large Scale Transcriptional Analysis: Cell Culture, DNA Arrays & Regulatory Network Analysis
	References

	Stem-Cell & Hematopoietic Tissue Engineering: Cell Biology, DNA Arrays & Models
	Project 1 Project 2
	Collaborators
	Hematopoiesis is the production of mature blood cells from stem cells in the bone marrow (BM). Stem and progenitor cells from the BM, umbilical-cord blood, or peripheral blood can be used to restore hematopoietic activity in patients whose marrow has been ablated by chemotherapy. Ex vivo expansion of hematopoietic stem and progenitor cells has the potential to enlarge the donor pool by reducing the required harvest size, to decrease variability in patient engraftment and increase the survival rate of patients. Because stem cells give rise to many cell types and "home" to the BM after manipulation outside the body, they are also attractive targets for gene therapy to treat a wide range of genetic disorders. The recently discovered plasticity of stem cells (e.g., from blood stem cells to brain cells, and from brain stem cells to blood cells), combined with new tools in molecular and cell biology of the genomic and post-genomic era, hold exceptional promise for large advances in both the biology and bioengineering of stem cells. A major limitation for stem-cell based therapies is the small number of stem cells available from the various tissue sources. As a result, extensive stem cell expansion will be required for most if not all applications. A related challenge that has proven even more difficult to overcome is the controlled expansion of stem cells without concomitant differentiation into more mature cell types - this process is often termed stem-cell self-renewal. The design and operation of culture systems is also a major challenge because stem-cell self-renewal and differentiation may be greatly altered by changes in the culture environment (such as pH, oxygen tension (pO₂), growth factor concentrations and presentation, use of accessory cells, and adhesion molecules). There are two major projects currently under investigation.

	Project 1
	Experimental evidence suggests that there exist oxygen tension (pO₂) and pH gradients in the BM hematopoietic compartment (BMHC). Our hypothesis is that such pO₂ and pH gradients are significant and play a key role in regulating the differentiation and proliferation of cells in the various hematopoietic lineages. This model is likely to have a major impact on the development of better chemotherapy and radiation treatments for cancer, as a well as on the design of devices and protocols for ex vivo expansion of hematopoietic cells for cell and gene therapies, and for the eventual production of mature hematopoietic cells for transfusion therapies. We have used model simulations to show the extent and magnitude of these gradients (Refs. 1&2), and experimental tools to show the importance of pO₂ and pH on the proliferation and differentiation of both the granulocytic (G)(Refs. 3, 4 & 16) and megakaryocytic (Mk) lineage (refs. 4 &5). Granulocytes are the white blood cells that fight bacterial infections, and megakaryocytes give rise to platelets, which are crucial for blood coagulation and trauma repair. Current work focuses on the understanding the large-scale transcriptional program of both G and Mk lineages using DNA arrays, Northern and Western analysis, immunofluorescence microscopy and computational methods.

	Project 2
	Stem cell division in culture is typically associated with differentiation into committed progenitor cells of the various lineages. It has been reported that ex vivo stem-cell expansion is increased by the presence of a stromal cell feeder layer, which replaces some of the functions of stromal cells in the BM. We investigate the hypothesis that immobilized growth factors (and notably stem-cell factor, SCF) result in enhanced ex vivo stem cell renewal. We will also develop mathematical models that will allow us to determine whether differences in stem-cell renewal with immobilized SCF can be explained by quantitative understanding of key cellular processes. Finally, modeling of the early stages of stem cell differentiation will be undertaken using a new model for early hematopoiesis.

	Collaborators
	W. M. Miller (Chemical Engineering Dept.) J. Winter (Hematology/Oncology, NU Weinberg School of Medicine) E. Eklund (Hematology/Oncology, NU Weinberg School of Medicine)
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	Metabolic Engineering, DNA Arrays and Functional Genomics of Solventogenic Clostridia
	Collaborators
	Production of solvents and other oxychemicals from renewable resources is now enjoying a new renaissance in view of recent major advances in genomics, Metabolic Engineering (ME) and high throughput technologies. My group in developing ME and genomic-based strategies in order to generate recombinant strains (Refs. 7, 8) of Clostridium acetobutylicum for applications in the production of solvents and other chemicals, biocatalysis, and bioremediation. A key and fast-evolving high throughput technology is transcriptional analysis using DNA arrays. Transcriptome analysis, as it has been termed, by DNA arrays, enables for the first time to examine the cell from a system's point of view, and to elucidate the interconnection between the various metabolic and regulatory networks and programs of the cell. This is the essence of what is termed functional genomics and requires substantial skills in bioinformatics. This capability, combined with flux analysis (Refs. 8, 9, 10) - another bioinformatics tool which has been recently redeveloped and advanced for this organism by our group - may eventually allow us to relate the genetic make up of the cell to the metabolic flux network and regulation used by the cell in response to various stimuli or imposed conditions. We use this approach to examine the large transcriptional programs of differentiation (sporulation, Ref. 17), product formation, response to toxic chemicals, and other stresses. Furthermore, design aspects of the antisense RNA (asRNA) technology -which has also been recently developed for this organism by our group (Ref. 11) - are examined in detail, and further advancements of this technology for the generation of overproducing strains will be pursued. We are also interested in understanding solvent (and other toxic chemical) tolerance of microorganisms, which is crucial for the production of chemicals, bioremediation, and whole-cell biocatalysis. Past efforts to produce tolerant strains have relied on selection under applied pressure and chemical mutagenesis, with some good results, but not consistently so. We desire to examine if ME and genomic approaches can be used to construct more tolerant strains for bioprocessing. The objective of this research is to identify genes that contribute to solvent tolerance and to use genetic modifications (involving these genes) to generate solvent tolerant strains. In view of the large number of possible genes that may be involved in determining solvent tolerance, we use DNA microarrays based on the recently published genome sequence of C. acetobutylicum. DNA microarrays were designed and constructed in our laboratory in order to examine the large-scale transcriptional program of the cells in response to various levels of butanol and other solvent challenges. Many genes belonging to several classes (molecular pumps, chaperonins (HSPs), primary metabolism, ATPases, sporulation, transcriptional regulators, carbohydrate metabolism) were identified as changing gene expression under solvent stress. Several of these genes are explored in ME studies.

	Collaborators
	G. Bennett (Biochemistry and Cell Biology, Rice University, Houston, TX) E. Weiss (Biochemistry, Molecular Biology & Cell Biology, NU)
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	T-Cell Based Cellular Immunotherapies & Large Scale Transcriptional Analysis: Cell Culture, DNA Arrays & Regulatory Network Analysis
	Cellular immunotherapy, whereby a patient is treated with large doses of autologous ex vivo-expanded immune cells in order to eradicate aberrant - such as malignant or virally infected- cells, offers an alternative treatment for such diseases. The effectiveness of these therapies is considered to be dose dependent, as patient survival increases proportionately with larger doses of effector cells (Ref. 12). As a result, treatment regimens require large numbers of cells, typically 10⁹ to 10¹¹ cells per patient, making expansion of the cells via ex vivo cell culture necessary. In expanding T cells for immunotherapy it is important not only to generate large numbers of cells, but also to ensure that the cell product is of high quality such that it will be biologically active upon transfusion back to the patient. Thus, optimization of the cell culture parameters used for expansion of these cells is a crucial issue for the success of cellular immunotherapy (Refs. 12-14). Some culture parameters, like oxygen tension (Refs. 12 and 15) and autologous plasma (Ref. 15) have profound (5 to 70-fold) effects on cell proliferation and differentiation. Understanding how such culture parameters affect T-cell metabolism, proliferation and differentiation at the cellular and molecular level is not only necessary for culture-protocol optimization but also important basic knowledge. Our work aims to understand the cellular and molecular basis of these events, which are likely due to alterations in a number of cellular networks and signal-transduction cascades. Candidates include the cell-cycle network, the apoptotic cascade, as well the glycolytic pathway. Thus, experimental techniques that allow one to undertake a systems approach examination of the cell (such as DNA arrays) are key tool sin this endeavor, coupled of course with established tools from molecular biology, biochemical engineering, and systems theory.
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	References
	1.	Chow, D, W. M. Miller and E. T. Papoutsakis, "Estimation of oxygen tension distributions in the bone marrow hematopoietic compartment. I. Krogh's model", Biophysical J. 81, 675-684 (2001).
	2.	Chow, D, W. M. Miller and E. T. Papoutsakis, "Estimation of oxygen tension distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models", Biophysical J. 81, 685-696 (2001).
	3.	Hevehan, D. L., Papoutsakis, E. T. & Miller W. M., "Physiologically significant effects of pH and oxygen tension on granulopoiesis", Exp. Hematol., 28: 267-275 (2000).
	4.	D. Hevehan, W. M. Miller, and E. T. Papoutsakis, "A Dynamic model of ex vivo granulocytic kinetics to examine the effects of pO₂, pH and IL-3", Exp. Hematol. 28:1016-1028 (2000).
	5.	Mostafa, S.S., Miller, W.M., and Papoutsakis, E.T. "Oxygen tension influences the differentiation, maturation and apoptosis of human megakaryocytes", Br. J. Haematol. 111: 879-889 (2000).
	6.	Mostafa, S.S., Papoutsakis, E.T., and Miller, W.M. "Oxygen tension modulates the expression of cytokine receptors, transcription factors and lineage-specific markers in cultured human megakaryocytes", Exper. Hematol. 29: 873-883 (2001).
	7.	Nair, R., Green E., Bennett, G. N. and Papoutsakis, E T. "Regulation of the sol locus genes for butanol and acetone production in Clostridium acetobutylicum ATCC 824 by a putative transcriptional repressor", J. Bacteriol. 181: 319-330 (1999).
	8.	Harris, L. M., Desai, R. P., Welker, N. E., Papoutsakis, E. T. "Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition?", Biotechnol. Bioeng., 67: 1-11 (2000).
	9.	Desai, R., Nielsen, L. K., and Papoutsakis, E. T. "Stoichiometric modeling of Clostridium acetobutylicum fermentations with nonlinear constraints", J. Biotechnol., 71: 191-205 (1999).
	10.	Desai, R. P., Harris, L. M., Welker, N. E., Papoutsakis, E. T. "Metabolic flux analysis elucidates the importance of the acid-formation pathway in regulating solvent production by Clostridium acetobutylicum ", Metabolic Engineering 1: 206-213 (1999).
	11.	Desai, R. P. and Papoutsakis, E. T., "Antisense RNA strategies for the metabolic engineering of Clostridium acetobutylicum", Appl. Environ. Microbiol., 65: 936-945 (1999).
	12.	Carswell, K. S., Weiss, J. W. and Papoutsakis, E. T. "Low oxygen tension enhances the stimulation and proliferation of human T lymphocytes", Cytotherapy, 2: 25-37 (2000).
	13.	Carswell, K. S., and Papoutsakis, E. T. "Culture of human T cells in stirred bioreactors for cellular immunotherapy applications: shear, proliferation, and the IL-2 receptor", Biotechnol. Bioeng. 68: 328-338 (2000).
	14.	Carswell, K. S., and Papoutsakis, E. T. "Extracellular pH affects the proliferation of cultured human T cells and their expression of the Interleukin 2 receptor", J. Immunotherapy, 23: 669-674 (2000).
	15.	Haddad, H. and Papoutsakis, E. T. "Low oxygen tension and autologous plasma increase T-cell proliferation in serum-free media", Cytotherapy 3: 435-447 (2001).
	16.	Hevehan, D. L., Miller, W. M. and Papoutsakis, E. T. "Differential expression and phosphorylation of distinct STAT3 proteins during granulocytic differentiation", Blood 99: 1627-1637 (2002).
	17.	Harris, L. M. , Welker, N. E. and Papoutsakis, E. T. "Northern, morphological and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824". J. Bacteriol. 184: 3586-3597 (2002).
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