bone marrow derived stem cells in normal physiology and in cancer. an introduction

Bone marrow derived stem cells in normal physiology and in cancer.

An introduction

Hill, J. M. et al. NEJM 2003;348:593-600

Association between Cardiovascular Risk Factors and Endothelial-Progenitor-Cell

Colony Counts

Survival analyzed for cardiovascular death compared to levels of CD34+, KDR+, circulating endothelial progenitor cells

Werner N, Kosiol S. et al. NEJM 2005;353:999-1007

Intra-coronary autologous bone marrow cell transfer after myocardial infarction. The BOOST randomised controlled trial.

Wollert KC, Meyer GP, et al. Lancet 2004; 364: 141-148

60 patients randomized 1:1

Given approximately 9x106 CD34+ cells via injection in coronary artery



4 of 5 outcome measure indicators showed no change.

REVIVAL2: Stem cell mobilization by granulocyte-colony stimulating factor for patients with acute myocardial infarction. A randomized, controlled trial.

114 patients randomized 1:1

Given G-CSF + standard care

10 fold increase in circulating endothelial cells after 1 week

Zohlnhoefer D, Ott I, et al. JAMA 2006; 295: 1003-1010

REVIVAL2: Stem cell mobilization by granulocyte-colony stimulating factor for patients with acute myocardial infarction. A randomized, controlled trial.

Zohlnhoefer D, Ott I, et al. JAMA 2006; 295: 1003-1010

Stem cells and differentiation in the hematopoietic lineage

Zipori D. Stem Cells 2005; 23: 719-726

“Stem state: Plasticity is essential, … self renewal and hierarchy are optional.”

Zipori D. Stem Cells 2005; 23: 719-726

Mezey, Vogelsang, et al. PNAS 2003; 100: 1364-1369

Transplanted bone marrow generates new neurons in human brains

Autopsy material was studied from 4 female patients who had bone marrow transplant from male donors for leukemia and died months to years after the transplant.

Each 6 micron section of brain was stained with 3 immunofluorescent reagents marking Y chromosome, neural differentiation, and cell nucleus.

Mezey, Vogelsang, et al. PNAS 2003; 100: 1364-1369

A 6-µm-thin section from somatosensory cortex of patient 2 demonstrates the presence of the Y chromosome depicted as red dots and viewed through a rhodamine filter. The immunostaining for the neuronal marker NeuN in green (B), and the UV filter shows all cell nuclei in blue after staining with 4',6-diamidino-2-phenylindole, a chromosomal stain (C). (D) The overlay of the three filters, where arrows point to cells that carry all markers, indicating that they derived from the donor bone marrow (Y chromosome positive) and bear the specific neuronal marker NeuN. Arrowheads point at nonneuronal donor-derived cells. (Scale bars, 10 µm.)

Liver from bone marrow in humans

Theise ND, Krause DS, et al. Hepatology 2000; 32: 11 - 16

Liver biopsies of male recipients of liver transplants from female donors

Slides examined and photographed with IHC to identify hepatocytes and cholangiocytes, then restained with FISH to identify X and Y chromosomes

Theise ND, Krause DS, et al. Hepatology 2000; 32: 11 - 16

Immuno for CK 8, 18, 19 Red = X chromosomeTurquoise = Y chromosomeBlue is DAPI

Liver from bone marrow in humans

Increased chimerism of bronchial and alveolar epithelium in human lung allografts undergoing chronic injury

Kleeberger W, Versmold A, et al. Am J Path 2003; 162: 1487 - 1494

Laser-capture microdissected samples of tissue from transplanted lung in gender-mismatched cases.

PCR used to quantitate X and Y chromosome to measure % chimerism.

Transplanted lung with chronic inflammation and squamous metaplasia

Recipient genotype by short tandem repeat PCR

Kleeberger W, Versmold A, et al. Am J Path 2003; 162: 1487 - 1494

Donor and recipient genotype detected

Transplanted lung with normal bronchial glands

Lesser degree of chimerism

Progenitor cells from bone marrow can migrate into blood vessels, liver, myocardium, and brain.

BMDC’s can differentiate and incorporate at site of implantation.

This process is more extensive if there is chronic inflammation at the site.

Homing and implantation may be controlled by expression of specific integrins and may require the the stimulus of VEGF.

This physiologic process has not yet been used therapeutically.

Biofluorescence in Aequorea victoria

Fluorescent proteins from Aequorea and Anthozoa do not require cofactors or ATP

Intrinsic luminescence is 1,000 fold greater than luciferin

Verkuosha, Lukyanov. Nature Biotechnology 2004; 22: 289-296

Fluorescent proteins can be transfected into a wide variety of tissues and organisms with genetic control of expression.

Verkuosha, Lukyanov. Nature Biotechnology 2004; 22: 289-296

Hoffman, R. Nature Reviews Cancer 2005; 5: 796-806

A metastasis in lung composed of cells from 2 different clones of fibrosarcoma

Lyden D, Hattori K, et al. Nature Medicine 2001; 7: 1194-1201

Id protein deficient mice are resistant to the growth of transplanted tumors

Susceptibility returns with transplantation of normal bone marrow or VEGF-mobilized circulating endothelial precursor cells

Id mouse model of CEP cells in tumor angiogenesis



Transplanted LacZ+ bone marrow cells forming a blood vessel in a tumor in an Id1+/-Id3-/- mouse


The effect of transplanting Id- animal with normal bone marrow is blocked if treated with antibody against VEGF receptor.




Tumor growth requires participation of endothelial precursor cells mobilized from the bone marrow by VEGF (vascular endothelial growth factor)

Jin, Aiyer, et al. J Clin Invest Mar 2006; 116: 652-662

Homing of bone-marrow derived cells in tumor angiogenesis

Sections of N202 breast cancer growing in a nu/nu mouse with injected with GFP+, CD34+ bone marrow cells

Jin, Aiyer, et al. J Clin Invest Mar 2006; 116: 652-662

Homing of bone-marrow derived cells in tumor angiogenesis

In this system, homing of bone marrow cells depends on expression of 41 integrin (VLA-4).

The bone marrow-derived cells differentiate into endothelial cells within the growing tumor.

Homing of bone marrow-derived precursors cells before tumor metastasis

Kaplan R N, Lyden D, et al. Nature Dec 2005; 438: 820-827



Lung following bone marrow transplant, before injection of melanoma cells

Lung day 14 after injection of melanoma cells

C57BL/6 ROSA26 eGFP

Cells express GFP and non-mammalian beta-galactosidase

Radiation

Bone marrow transplant with male donor, female recipient

C57BL/6 mouse transplanted with GFP+, -gal+ bone marrow or wild-type marrow

Infected with Helicobacter

Gastric muscosa assayed for GFP, beta-galactosidase

Immunohistochemical stain for beta-galactosidase in in dysplastic gastric glands with chronic Helicobacter infection. In C note beta-galactosidase in cytoplasm of adipocyte.

Li, Houghton, et al. W. J. Gastroenterology. 2006; 12(3): 363-371

Li, Houghton, et al. W. J. Gastroenterology. 2006; 12(3): 363-371

Houghton, Stoicov, et al. Science. 2004; 306: 1568-71

Gastric glands with intra-epithelial neoplasia with immunofluorescence for cytokeratin (green) and beta-galactosidase (red).

Wild-type marrow ROSA26,eGFP marrow

Houghton, Stoicov, et al. Science. 2004; 306: 1568-71

Tumor cells in gastric carcinoma (black nuclei) with Y chromosome by FISH (green) in cells with cytokeratin immunofluorescence (red).

» Stem cells exist in the normal bone marrow that can engraft in endothelial, mesenchymal, and epithelial tissues.

» The cytokine and integrin signals that control mobilization, tissue homing, engraftment, proliferation, and differentiation are not fully known.

» Engraftment of normal tissues by bone marrow derived stem cells may be permissive or required for tumor metastasis.

» Sites of engraftment following chronic inflammation may be vulnerable to malignant transformation.

» Tumors in solid tumors may arise in stem cells in tissue derived from bone marrow.

Thank you.

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bone marrow derived stem cells in normal physiology and in cancer. an introduction

Documents

bone marrow derived

introduction slide

cell colony counts slide

bone marrow transplant

endothelial cells

coronary artery slide

transplanted bone marrow

stem cell mobilization