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During progression to type 1 diabetes, insulin-producing β-cells are lost through an autoimmune attack resulting in unrestrained glucagon expression and secretion, activation of glycogenolysis, and escalating hyperglycemia. We recently identified a protein, designated islet homeostasis protein (IHoP), which specifically co-localizes within glucagon-positive α-cells and is overexpressed in the islets of both post-onset non-obese diabetic (NOD) mice and type 1 diabetes patients. Here we report that in the αTC1.9 mouse α-cell line, IHoP was released in response to high-glucose challenge and was found to regulate secretion of glucagon. We also show that in NOD mice with diabetes, major histocompatibility complex class II was upregulated in islets. In addition hyperglycemia was modulated in NOD mice via suppression of IHoP utilizing small interfering RNA (IHoP-siRNA) constructs/approaches. Suppression of IHoP in the pre-diabetes setting maintained normoglycemia, glyconeolysis, and fostered β-cell restoration in NOD mice 35 weeks post treatment. Furthermore, we performed adoptive transfer experiments using splenocytes from IHoP-siRNA-treated NOD/ShiLtJ mice, which thwarted the development of hyperglycemia and the extent of insulitis seen in recipient mice. Last, IHoP can be detected in the serum of human type 1 diabetes patients and could potentially serve as an early novel biomarker for type 1 diabetes in patients.

During progression to type 1 diabetes, insulin-producing β-cells are lost through an autoimmune attack resulting in unrestrained glucagon expression and secretion, activation of glycogenolysis, and escalating hyperglycemia. We recently identified a protein, designated islet homeostasis protein (IHoP), which specifically co-localizes within glucagon-positive α-cells and is overexpressed in the islets of both post-onset non-obese diabetic (NOD) mice and type 1 diabetes patients. Here we report that in the αTC1.9 mouse α-cell line, IHoP was released in response to high-glucose challenge and was found to regulate secretion of glucagon. We also show that in NOD mice with diabetes, major histocompatibility complex class II was upregulated in islets. In addition hyperglycemia was modulated in NOD mice via suppression of IHoP utilizing small interfering RNA (IHoP-siRNA) constructs/approaches. Suppression of IHoP in the pre-diabetes setting maintained normoglycemia, glyconeolysis, and fostered β-cell restoration in NOD mice 35 weeks post treatment. Furthermore, we performed adoptive transfer experiments using splenocytes from IHoP-siRNA-treated NOD/ShiLtJ mice, which thwarted the development of hyperglycemia and the extent of insulitis seen in recipient mice. Last, IHoP can be detected in the serum of human type 1 diabetes patients and could potentially serve as an early novel biomarker for type 1 diabetes in patients.

This report evaluates the spatial profile of blood vessel fragments (BVFs) and CD34+ and CD117+ hematopoietic stem and progenitor cells (HSPCs) in human cancellous bone. Bone specimens were sectioned, immunostained (anti-CD34 and anti-CD117), and digitally imaged. Immunoreactive cells and vessels were then optically and morphometrically identified and labeled on the corresponding digital image. The distance of each BVF, or CD34+ or CD117+ HSPC to the nearest trabecular surface was measured and binned in 50-μm increments. The relative concentration of HSPCs and BVFs within cancellous marrow was observed to diminish with increasing distance in the marrow space. On average, 50% of the CD34+ HSPC population, 60% of the CD117+ HSPC population, and 72% of the BVFs were found within 100 μm of the bone surfaces. HSPCs were also found to exist in close proximity to BVFs, which supports the notion of a shared HSPC and vessel spatial niche.

This report evaluates the spatial profile of blood vessel fragments (BVFs) and CD34+and CD117+hematopoietic stem and progenitor cells (HSPCs) in human cancellous bone. Bone specimens were sectioned, immunostained (anti-CD34 and anti-CD117), and digitally imaged. Immunoreactive cells and vessels were then optically and morphometrically identified and labeled on the corresponding digital image. The distance of each BVF, or CD34+or CD117+HSPC to the nearest trabecular surface was measured and binned in 50-μm increments. The relative concentration of HSPCs and BVFs within cancellous marrow was observed to diminish with increasing distance in the marrow space. On average, 50% of the CD34+HSPC population, 60% of the CD117+HSPC population, and 72% of the BVFs were found within 100 μm of the bone surfaces. HSPCs were also found to exist in close proximity to BVFs, which supports the notion of a shared HSPC and vessel spatial niche.

Reports of neural transdifferentiation of mesenchymal stem cells (MSCs) suggest the possibility that these cells may serve as a source for stem cell–based regenerative medicine to treat neurological disorders. However, some recent studies controvert previous reports of MSC neurogenecity. In the current study, we evaluate the neural differentiation potential of mouse bone marrow–derived MSCs. Surprisingly, we found that MSCs spontaneously express certain neuronal phenotype markers in culture, in the absence of specialized induction reagents. A previously published neural induction protocol that elevates cytoplasmic cyclic AMP does not upregulate neuron‐specific protein expression significantly in MSCs but does significantly increase expression of the astrocyte‐specific glial fibrillary acidic protein. Finally, when grafted into the lateral ventricles of neonatal mouse brain, MSCs migrate extensively and differentiate into olfactory bulb granule cells and periventricular astrocytes, without evidence of cell fusion. These results indicate that MSCs may be “primed” toward a neural fate by the constitutive expression of neuronal antigens and that they seem to respond with an appropriate neural pattern of differentiation when exposed to the environment of the developing brain.

Stromal derived factor-1 alpha (SDF-1α) and its receptor CXCR4 have been shown to play a role in the systematic movement of hematopoietic stem cells (HSC) in the fetal and adult stages of hematopoiesis. Under certain physiological conditions liver oval cells can participate in the regeneration of the liver. We have shown that a percentage of oval cells are of hematopoietic origin. Others have shown that bone marrow derived stem cells can participate in liver regeneration as well. In this study we examined the role of SDF-1α and its receptor CXCR4 as a possible mechanism for oval cell activation in oval cell aided liver regeneration. In massive liver injury models where oval cell repair is involved hepatocytes up-regulate the expression of SDF-1α, a potent chemoattractant for hematopoietic cells. However, when moderate liver injury occurs, proliferation of resident hepatocytes repairs the injury. Under these conditions SDF-1α expression is not up-regulated and oval cells are not activated in the liver. In addition, we show that oval cells express CXCR4, the only known receptor for SDF-1α. Lastly, in vitro chemotaxis assays demonstrated that oval cells migrate along a SDF-1α gradient which suggests that the SDF-1α/CXCR4 interaction is a mechanism by which the oval cell compartment could be activated and possibly recruit a second wave of bone marrow stem cells to the injured liver. In conclusion, these experiments begin to shed light on a possible mechanism, which may someday lead to a better understanding of the hepatic and hematopoietic interaction in oval cell aided liver regeneration.

The murine adult hematopoietic stem cell is able to function as a hemangioblast, contributing both to blood reconstitution and to blood vessel repair in response to ischemic injury. We developed a novel mouse xenotransplantation model of retinal neovascularization to test human hematopoietic cell plasticity. Immunocompromised nonobese diabetic (NOD)/scid mice underwent myeloablative conditioning and transplantation with human CD34+ umbilical cord blood. After multilineage reconstitution was established, retinal ischemia was induced to promote neovascularization. Our results demonstrate human retinal neovascularization, thus revealing the functional hemangioblast activity of human hematopoietic cells.

The murine adult hematopoietic stem cell is able to function as a hemangioblast, contributing both to blood reconstitution and to blood vessel repair in response to ischemic injury. We developed a novel mouse xenotransplantation model of retinal neovascularization to test human hematopoietic cell plasticity. Immunocompromised nonobese diabetic (NOD)/scidmice underwent myeloablative conditioning and transplantation with human CD34+umbilical cord blood. After multilineage reconstitution was established, retinal ischemia was induced to promote neovascularization. Our results demonstrate human retinal neovascularization, thus revealing the functional hemangioblast activity of human hematopoietic cells.

Blood vessel development is essential to cancer growth and metastasis. Based on our recent findings that the hematopoietic stem cell (HSC) provides functional hemangioblast activity in repairing the ischemic retina, we questioned if the HSC also acts as a pathologic hemangioblast - contributing to tumor vasculogenesis. Using a transplant model of green fluorescent protein (GFP) marrow into wild-type C57BL/6 mice, we injected lung cancer (LLC) and allowed the tumor to grow for 14 days. All tumor specimens demonstrated tumor vessels with marrow-derived endothelial cells. Approximately 25% of tumor vessels contained marrow-derived endothelial cells as demonstrated by GFP, CD31 and vWF expression. Confocal microscopy was used to identify true marrow-derived endothelial cells lining the vascular lumen versus marrow-derived pericytes juxtaposed to endothelial cells. We further questioned whether the tumor neovasculogenesis is from a clonal, self-renewing HSC. Lung cancers grown in recipients of single cell and serially transplanted hematopoietic stem cells (n=9) demonstrate clonal, donor-derived endothelial cells in 5% of tumor vasculature, matching hematopoietic engraftment. Our results indicate that the self-renewing, clonal adult hematopoietic stem cell exhibits pathologic hemangioblast activity, capable of producing both blood and blood vessels within tumors. Given that the HSC and its EPC progeny can contribute to tumor vasculogenesis, we further hypothesized that factors that affect leukocyte trafficking likely affect the pathologic hemangioblast activity of HSC. Thus, we made slight modifications to our transplant and tumor inoculation model by administering GCSF and SCF to mobilize marrow derived EPC. Over the ensuing 14 days, the tumors in the cytokine treated group grew at a much faster rate and to a much larger size than tumors in the control mice. After 14 days of cytokine treatment and tumor growth, microvessel density was not different between cytokine treated mice (n=4) and control mice (n=4); however, marrow-derived tumor vasculogenesis was markedly elevated in the cytokine treated compared to controls (63% vs. 26%). In conclusion, the HSC contributes to blood vessels within lung cancer and strategies targeting HSC/EPC mobilization (such as during the recovery phase of chemotherapy) potentially represent excellent ant-neoplastic opportunities.

Human marrow has shown potential to contribute to organ various organs. We previously demonstrated marrow contribution to the brain in humans, without evidence for fusion. Given the observations of marrow contributing to the other end-organs, we questioned whether marrow also repair endocrine glands like the thyroid and adrenal glands. To address this question, we identified thyroid (n=3) and adrenal tissues (n=3) obtained from women who received hematopoietic cell transplantation from male donors. Thyroid tissues were stained for thyroid peroxidase (TPO), CD45, and XY FISH. Adrenal tissues were stained for synaptophysin, CD45, and XY FISH. Analysis of 450 thyroid follicles (150 per patient) found 2% to 5% of follicles containing a male/marrow-derived thyroid follicle demonstrating follicular orientation, TPO expression, lacking CD45, and containing a Y chromosome. The median number of male/marrow-derived cells in follicles was 1. None of the male/marrow-derived cells demonstrated a fusion karyotype (XXY or XXXY). In contrast, adrenal tissues analyzed in the women demonstrated no male cells throughout all layers. In conclusion, our results show that human marrow can contribute to thyroid follicles and express thyroid proteins, however at a modest level. Furthermore, adrenal tissues in these women demonstrated no evidence of marrow contribution. These results expose a potential strategy of thyroid repair by illuminating the potential of marrow contribution to thyroid epithelia.

The hematopoietic stem cell (HSC) is capable of contributing to vasculogenesis in response to ischemic injury. We have previously demonstrated that adult hemangioblast activity also exists within the capabilities of human hematopoietic stem/progenitor cells (HSC/HPC). However, we question the clinical relevance of adult hemangioblast activity. Thus, to address this question, we sought to analyze the marrow contribution to blood vessels within damaged vascular beds. Given the nephrotoxic side effects of chemotherapy and hematopoietic cell transplantation regimens, we identified kidney tissues from women (n=4) who received hematopoietic cell transplantations from male donors. Kidney tissues were stained for CD31, CD45, and XY FISH. One hundred forty vascular fields were analyzed. Fields were divided into three zones based on physiologic functions: glomerular, arteriolar (afferent & efferent), and medullary. In ever zone we identified male/marrow-derived endothelial cells contributing to kidney vasculature. However, each zone differed as to the degree of male/marrow-derived vascular contribution. In the glomerular regions, 36% of glomeruli contained one or more marrow-derived endothelial cells, as determined by orientation at the lumen edge, CD31 surface expression, and Y chromosome staining. In contrast, only 4% of arterioles contained male/marrow-derived endothelial cells. In the medulla, 9% of blood vessels contained male/marrow-derived blood vessels. The median number of marrow-derived endothelial cells in all regions was 1 male cell per blood vessel in field. In conclusion, human marrow can contribute modestly to kidney vasculature, especially in glomeruli. These results expose a potential strategy of kidney vessel repair by illuminating marrow-derived vasculogenesis within the kidney.

Leukemia stem cell activity has been extensively studied and has been approached mainly in terms of self-renewal within the hematopoietic compartment. However, given the recent findings that the adult hematopoietic stem cell can provide hemangioblast activity - giving rise to both blood and blood vessels - we questioned whether leukemia also exhibits hemangioblast activity. To test this hypothesis we established a xenotransplant model whereby K562 human myeloid leukemia cells suspended in Matrigel are injected into sublethally irradiated NOD/scid mice. Leukemia loaded Matrigel plugs reliably grew in size in all (n=6) immunocompromised animals. After four weeks of leukemia growth the Matrigel plugs were excised and blood vessels evaluated. Out of six animals, two animals demonstrated human endothelial cells within the Matrigel plug, confirmed by LAMP-1 staining. Interestingly, six out of six animals demonstrated mouse endothelial cells within the Matrigel. This in vivo model demonstrates that human myeloid leukemia can provide hemangioblast activity. Moreover, it appears that within this Matrigel xenotransplant model, host blood vessels are also recruited. The significance of these results highlights the importance in recognizing the hemangioblast potential of leukemia stem cells. Ultimately, complete eradication of leukemia may depend on targeting leukemia-derived blood vessels in addition to cytotoxic therapy directed at the hematopoietic compartment.

Megakaryocytes, which give rise to platelets, constitute a small population of cells. However, molecular aspects of megakaryocyte development are poorly understood. MafG is the small subunit of the transcription factor, NF-E2, which plays a major role in terminal megakaryocyte differentiation and platelet release. We sought to determine the effect of impaired platelet release due to targeted ablation of the mafG gene following a hematopoietic stem cell transplant on engraftment kinetics. We performed three types transplants:C57Bl/6 Ly5.1 murine bone marrow (mafGWT/WT bm) into lethally irradiated mafG null hosts expressing green fluorescent protein, (GFP),mafG-null/GFP bm into lethally irradiated C57BL/6 mafGWT/WThosts, andmafGWT/WT GFP bm into lethally irradiated C57BL/6 mafGWT/WT hosts. We measured peripheral cell counts, the presence of circulating donor-derived cells by flow cytometry, changes in the cellularity of the bone marrow and splenic weight, and megakaryocyte size and concentration on day 5, 7, 14, and 28 post transplant. We found:Platelet engraftment kinetics differed when mafG null animals served as donors or recipients. Specifically, platelets reached their lowest point at day 7 when mafGWT/WT or mafG null bm was transplanted into wildtype hosts. When mafGWT/WT bm was transplanted into mafG null hosts, platelets reached a low point on day 5.MafG null hosts transplant with mafGWT/WT bm had 5-fold lower circulating platelet levels than mafGWT/WT hosts at day 5.When mafGWT/WT served as donors, circulating platelets began to increase after day 7, while animals receiving mafG null donor cells had platelet counts that continued to diminish.Splenic hematopoiesis was markedly decreased when mafGWT/WT bm was transplanted into mafG null hosts. In contrast, when mafG null bm was transplanted into mafGWT/WT hosts, splenic hematopoiesis was increased 2.5 fold on day 28 compared to animals receiving mafGWT/WTdonor cells.Mortality rate was much higher in mafG null hosts.Leukocyte engraftment was delayed when mafGWT/WT bm was transplanted into mafG null hosts. These results suggest that host factors determine the timing of the platelet nadir following bone marrow transplant. Furthermore, the mafG protein, contributes in previously unrecognized ways to splenic hematopoiesis and successful engraftment, possibly through its function in promoting megakaryocyte development and platelet release.

Leukemia stem cell activity has been extensively studied and has been approached mainly in terms of self-renewal within the hematopoietic compartment. However, given the recent findings that the adult hematopoietic stem cell can provide hemangioblast activity - giving rise to both blood and blood vessels - we questioned whether leukemia also exhibits hemangioblast activity. To test this hypothesis we established a xenotransplant model whereby K562 human myeloid leukemia cells suspended in Matrigel are injected into sublethally irradiated NOD/scid mice. Leukemia loaded Matrigel plugs reliably grew in size in all (n=6) immunocompromised animals. After four weeks of leukemia growth the Matrigel plugs were excised and blood vessels evaluated. Out of six animals, two animals demonstrated human endothelial cells within the Matrigel plug, confirmed by LAMP-1 staining. Interestingly, six out of six animals demonstrated mouse endothelial cells within the Matrigel. This in vivo model demonstrates that human myeloid leukemia can provide hemangioblast activity. Moreover, it appears that within this Matrigel xenotransplant model, host blood vessels are also recruited. The significance of these results highlights the importance in recognizing the hemangioblast potential of leukemia stem cells. Ultimately, complete eradication of leukemia may depend on targeting leukemia-derived blood vessels in addition to cytotoxic therapy directed at the hematopoietic compartment.

Blood vessel development is essential to cancer growth and metastasis. Based on our recent findings that the hematopoietic stem cell (HSC) provides functional hemangioblast activity in repairing the ischemic retina, we questioned if the HSC also acts as a pathologic hemangioblast - contributing to tumor vasculogenesis. Using a transplant model of green fluorescent protein (GFP) marrow into wild-type C57BL/6 mice, we injected lung cancer (LLC) and allowed the tumor to grow for 14 days. All tumor specimens demonstrated tumor vessels with marrow-derived endothelial cells. Approximately 25% of tumor vessels contained marrow-derived endothelial cells as demonstrated by GFP, CD31 and vWF expression. Confocal microscopy was used to identify true marrow-derived endothelial cells lining the vascular lumen versus marrow-derived pericytes juxtaposed to endothelial cells. We further questioned whether the tumor neovasculogenesis is from a clonal, self-renewing HSC. Lung cancers grown in recipients of single cell and serially transplanted hematopoietic stem cells (n=9) demonstrate clonal, donor-derived endothelial cells in 5% of tumor vasculature, matching hematopoietic engraftment. Our results indicate that the self-renewing, clonal adult hematopoietic stem cell exhibits pathologic hemangioblast activity, capable of producing both blood and blood vessels within tumors. Given that the HSC and its EPC progeny can contribute to tumor vasculogenesis, we further hypothesized that factors that affect leukocyte trafficking likely affect the pathologic hemangioblast activity of HSC. Thus, we made slight modifications to our transplant and tumor inoculation model by administering GCSF and SCF to mobilize marrow derived EPC. Over the ensuing 14 days, the tumors in the cytokine treated group grew at a much faster rate and to a much larger size than tumors in the control mice. After 14 days of cytokine treatment and tumor growth, microvessel density was not different between cytokine treated mice (n=4) and control mice (n=4); however, marrow-derived tumor vasculogenesis was markedly elevated in the cytokine treated compared to controls (63% vs. 26%). In conclusion, the HSC contributes to blood vessels within lung cancer and strategies targeting HSC/EPC mobilization (such as during the recovery phase of chemotherapy) potentially represent excellent ant-neoplastic opportunities.

Human marrow has shown potential to contribute to organ various organs. We previously demonstrated marrow contribution to the brain in humans, without evidence for fusion. Given the observations of marrow contributing to the other end-organs, we questioned whether marrow also repair endocrine glands like the thyroid and adrenal glands. To address this question, we identified thyroid (n=3) and adrenal tissues (n=3) obtained from women who received hematopoietic cell transplantation from male donors. Thyroid tissues were stained for thyroid peroxidase (TPO), CD45, and XY FISH. Adrenal tissues were stained for synaptophysin, CD45, and XY FISH. Analysis of 450 thyroid follicles (150 per patient) found 2% to 5% of follicles containing a male/marrow-derived thyroid follicle demonstrating follicular orientation, TPO expression, lacking CD45, and containing a Y chromosome. The median number of male/marrow-derived cells in follicles was 1. None of the male/marrow-derived cells demonstrated a fusion karyotype (XXY or XXXY). In contrast, adrenal tissues analyzed in the women demonstrated no male cells throughout all layers. In conclusion, our results show that human marrow can contribute to thyroid follicles and express thyroid proteins, however at a modest level. Furthermore, adrenal tissues in these women demonstrated no evidence of marrow contribution. These results expose a potential strategy of thyroid repair by illuminating the potential of marrow contribution to thyroid epithelia.

The hematopoietic stem cell (HSC) is capable of contributing to vasculogenesis in response to ischemic injury. We have previously demonstrated that adult hemangioblast activity also exists within the capabilities of human hematopoietic stem/progenitor cells (HSC/HPC). However, we question the clinical relevance of adult hemangioblast activity. Thus, to address this question, we sought to analyze the marrow contribution to blood vessels within damaged vascular beds. Given the nephrotoxic side effects of chemotherapy and hematopoietic cell transplantation regimens, we identified kidney tissues from women (n=4) who received hematopoietic cell transplantations from male donors. Kidney tissues were stained for CD31, CD45, and XY FISH. One hundred forty vascular fields were analyzed. Fields were divided into three zones based on physiologic functions: glomerular, arteriolar (afferent & efferent), and medullary. In ever zone we identified male/marrow-derived endothelial cells contributing to kidney vasculature. However, each zone differed as to the degree of male/marrow-derived vascular contribution. In the glomerular regions, 36% of glomeruli contained one or more marrow-derived endothelial cells, as determined by orientation at the lumen edge, CD31 surface expression, and Y chromosome staining. In contrast, only 4% of arterioles contained male/marrow-derived endothelial cells. In the medulla, 9% of blood vessels contained male/marrow-derived blood vessels. The median number of marrow-derived endothelial cells in all regions was 1 male cell per blood vessel in field. In conclusion, human marrow can contribute modestly to kidney vasculature, especially in glomeruli. These results expose a potential strategy of kidney vessel repair by illuminating marrow-derived vasculogenesis within the kidney.

Megakaryocytes, which give rise to platelets, constitute a small population of cells. However, molecular aspects of megakaryocyte development are poorly understood. MafG is the small subunit of the transcription factor, NF-E2, which plays a major role in terminal megakaryocyte differentiation and platelet release. We sought to determine the effect of impaired platelet release due to targeted ablation of the mafG gene following a hematopoietic stem cell transplant on engraftment kinetics. We performed three types transplants:C57Bl/6 Ly5.1 murine bone marrow (mafGWT/WTbm) into lethally irradiated mafG null hosts expressing green fluorescent protein, (GFP),mafG-null/GFP bm into lethally irradiated C57BL/6 mafGWT/WThosts, andmafGWT/WTGFP bm into lethally irradiated C57BL/6 mafGWT/WThosts.

Marrow sinusoidal capillaries provide a niche for megakaryocyte progenitors and possibly hematopoietic stem cells. We sought to determine the fate of host sinusoidal capillaries during marrow transplant. We transplanted whole bone marrow, 2000 sorted Linneg Sca-1pos c-kitpos (SKL) hematopoietic stem and progenitor cells, or single SKL cells (along with rescue marrow) from male mice expressing green fluorescent protein into lethally irradiated female C57/BL6 hosts. We used green fluorescence and the presence of the Y-chromosome to identify donor-derived cells. Sinusoidal engraftment was studied from day 5 up to 1 year post-transplant and in secondary transplants. We identified numerous donor-derived cells based on green fluorescence or presence of the Y chromosome lining the sinusoids, but it was unclear whether these cells were vascular or hematopoietic. Furthermore, we were unable to demonstrate expression of von Willebrand factor, CD31 and MECA-32 using immunohistochemistry in healthy sinusoids. We used two methods to definitively identify donor-derived endothelial cells. First, we identified these cells based on location, characteristic shape and nuclei on H&E stained sections. Serial high power fields were photographed and then the same sections were stained with X and Y FISH probes. Second, we identified donor endothelial cells based on uptake of intravenously injected Ac-LDL which was endocytosed predominantly by sinusoidal endothelial cells four hours after injection. Flushed femoral bone marrow was treated with dispase and collagenase, and disaggregated cells were lineage depleted with a standard lineage cocktail containing Mac-1 antibody. Cytospins were photographed, Ac-LDL staining cells identified, and the same cytospins were stained with X and Y FISH probes. We counted numerous donor-derived endothelial cells whether 2X106 whole bone marrow (n=6 ), 2X104 SKL cells (n=6), or single SKL cells (n=2) were transplanted. These donor-derived endothelial cells were functional based on their ability to uptake DiI AcLDL. These cells appeared adjacent to mature, donor-derived megakaryocytes, suggesting function as a niche for megakaryocyte progenitors. Furthermore, donor-derived endothelial cells were present at levels similar to hematopoietic engraftment in every animal analyzed, suggesting robust levels of repair. Single cell transplants were assessed at seven and nine months, and in secondary transplants, establishing that these cells self-renew. To determine the mechanism whereby HSC’s repair the bone marrow sinusoids, we measured the expression of stromal derived factor-1 (SDF-1) at various time points during the first two weeks post-transplant by semi-quantitative RT-PCR, ELISA, and immunohistochemistry. SDF-1 expression peaked 3 days post-transplant and was expressed primarily by damaged blood vessels. The temporal and spatial pattern of engraftment matched expression patterns of SDF-1. This study demonstrates repair of host sinusoids by donor-derived endothelial cells following transplant, with levels of reconstitution similar to that of the hematopoietic system. This repair may be mediated by expression of SDF-1 by damaged marrow vasculature. Repair of sinusoidal capillaries may be a primary role of the HSC during transplant that is necessary for successful hematopoietic engraftment.

Marrow sinusoidal capillaries provide a niche for megakaryocyte progenitors and possibly hematopoietic stem cells. We sought to determine the fate of host sinusoidal capillaries during marrow transplant. We transplanted whole bone marrow, 2000 sorted LinnegSca-1posc-kitpos(SKL) hematopoietic stem and progenitor cells, or single SKL cells (along with rescue marrow) from male mice expressing green fluorescent protein into lethally irradiated female C57/BL6 hosts. We used green fluorescence and the presence of the Y-chromosome to identify donor-derived cells. Sinusoidal engraftment was studied from day 5 up to 1 year post-transplant and in secondary transplants. We identified numerous donor-derived cells based on green fluorescence or presence of the Y chromosome lining the sinusoids, but it was unclear whether these cells were vascular or hematopoietic. Furthermore, we were unable to demonstrate expression of von Willebrand factor, CD31 and MECA-32 using immunohistochemistry in healthy sinusoids. We used two methods to definitively identify donor-derived endothelial cells. First, we identified these cells based on location, characteristic shape and nuclei on H&E stained sections. Serial high power fields were photographed and then the same sections were stained with X and Y FISH probes. Second, we identified donor endothelial cells based on uptake of intravenously injected Ac-LDL which was endocytosed predominantly by sinusoidal endothelial cells four hours after injection. Flushed femoral bone marrow was treated with dispase and collagenase, and disaggregated cells were lineage depleted with a standard lineage cocktail containing Mac-1 antibody. Cytospins were photographed, Ac-LDL staining cells identified, and the same cytospins were stained with X and Y FISH probes. We counted numerous donor-derived endothelial cells whether 2X106whole bone marrow (n=6 ), 2X104SKL cells (n=6), or single SKL cells (n=2) were transplanted. These donor-derived endothelial cells were functional based on their ability to uptake DiI AcLDL. These cells appeared adjacent to mature, donor-derived megakaryocytes, suggesting function as a niche for megakaryocyte progenitors. Furthermore, donor-derived endothelial cells were present at levels similar to hematopoietic engraftment in every animal analyzed, suggesting robust levels of repair. Single cell transplants were assessed at seven and nine months, and in secondary transplants, establishing that these cells self-renew. To determine the mechanism whereby HSC's repair the bone marrow sinusoids, we measured the expression of stromal derived factor-1 (SDF-1) at various time points during the first two weeks post-transplant by semi-quantitative RT-PCR, ELISA, and immunohistochemistry. SDF-1 expression peaked 3 days post-transplant and was expressed primarily by damaged blood vessels. The temporal and spatial pattern of engraftment matched expression patterns of SDF-1. This study demonstrates repair of host sinusoids by donor-derived endothelial cells following transplant, with levels of reconstitution similar to that of the hematopoietic system. This repair may be mediated by expression of SDF-1 by damaged marrow vasculature. Repair of sinusoidal capillaries may be a primary role of the HSC during transplant that is necessary for successful hematopoietic engraftment.

Bone marrow contains stem cells that can engraft in various distant organs. These observations prompted us to investigate the cellular origin of post-transplant solid tumor neoplasias found in women having received a bone marrow transplant at our facility. In women having received bone marrow transplantation from male siblings we found colonic adenomas post-transplant. Presence of donor-derived cells were examined using a combination of immunohistochemistry for cells expressing the pan-leukocyte antigen, CD45, and the colonocyte antigen, cytokeratin 20, in conjunction with fluorescent in situ hybridization for X and Y chromosomes. Confocal microscopy was used to evaluate for evidence of cell fusion. In these cases, recipients of gender-mismatched transplants showed evidence of Y-chromosome positive, cytokeratin positive cells within the adenomas. The male cells accounted for 1 to 4% of the adenoma cells in histologic section. Confocal microscopy permitted evaluation of entire nuclei (Z-stack steps of 0.25 microns) and demonstrated no male cell with more than one X chromosome. A total of 40 male cells expressing cytokeratin were found throughout the adenomas. When considering how bone marrow derived cells incorporate into colonic adenomas, two possibilities should be regarded. First, bone marrow derived cells may migrate to neoplasias due to upregulation of tumor growth factors, which also act as inflammatory cytokines and chemokines, such as stromal derived factor 1 (SDF-1). Up- or down-regulating SDF-1 has profound effects on recruiting circulating bone marrow stem/progenitor cells for engraftment as end-organ epithelia. SDF-1 staining of the colonic adenomas found in our patients demonstrated intense, patchy SDF-1 expression compared to normal colonic epithelia. Second, roving donor-derived bone marrow cells may have engulfed the recipient adenoma cells. A donor scavenger cell could have phagocytosed an apoptotic adenoma cell. In these situations the fusion of nuclear material should result in a multiplicity of sex chromosomes (XXY or XXXY). However, as noted, the donor-derived epithelial cells within the adenomas demonstrated no extra X chromosomes. In the liver, where cell fusion has been demonstrated in severe disease stress states, it has been postulated that 28% of donor-derived hepatocytes are due to reduction division, resulting in diploid daughter cells. Based on the probability of binomial distribution, the chance that we would find 40 out of 40 diploid donor-derived epithelial cells amidst a background fusion resolution rate of 28% is one in 1x10e22. Thus, direct differentiation, rather then absolutely complete and perfect resolution of every fusion event is the most likely explanation of our current findings.

Bone marrow contains stem cells that can engraft in various distant organs. These observations prompted us to investigate the cellular origin of post-transplant solid tumor neoplasias found in women having received a bone marrow transplant at our facility. In women having received bone marrow transplantation from male siblings we found colonic adenomas post-transplant. Presence of donor-derived cells were examined using a combination of immunohistochemistry for cells expressing the pan-leukocyte antigen, CD45, and the colonocyte antigen, cytokeratin 20, in conjunction with fluorescent in situ hybridization for X and Y chromosomes. Confocal microscopy was used to evaluate for evidence of cell fusion. In these cases, recipients of gender-mismatched transplants showed evidence of Y-chromosome positive, cytokeratin positive cells within the adenomas. The male cells accounted for 1 to 4% of the adenoma cells in histologic section. Confocal microscopy permitted evaluation of entire nuclei (Z-stack steps of 0.25 microns) and demonstrated no male cell with more than one X chromosome. A total of 40 male cells expressing cytokeratin were found throughout the adenomas. When considering how bone marrow derived cells incorporate into colonic adenomas, two possibilities should be regarded. First, bone marrow derived cells may migrate to neoplasias due to upregulation of tumor growth factors, which also act as inflammatory cytokines and chemokines, such as stromal derived factor 1 (SDF-1). Up- or down-regulating SDF-1 has profound effects on recruiting circulating bone marrow stem/progenitor cells for engraftment as end-organ epithelia. SDF-1 staining of the colonic adenomas found in our patients demonstrated intense, patchy SDF-1 expression compared to normal colonic epithelia. Second, roving donor-derived bone marrow cells may have engulfed the recipient adenoma cells. A donor scavenger cell could have phagocytosed an apoptotic adenoma cell. In these situations the fusion of nuclear material should result in a multiplicity of sex chromosomes (XXY or XXXY). However, as noted, the donor-derived epithelial cells within the adenomas demonstrated no extra X chromosomes. In the liver, where cell fusion has been demonstrated in severe disease stress states, it has been postulated that 28% of donor-derived hepatocytes are due to reduction division, resulting in diploid daughter cells. Based on the probability of binomial distribution, the chance that we would find 40 out of 40 diploid donor-derived epithelial cells amidst a background fusion resolution rate of 28% is one in 1x10e22. Thus, direct differentiation, rather then absolutely complete and perfect resolution of every fusion event is the most likely explanation of our current findings.