京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22319号 / 医博第4560号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 濵﨑 洋子, 教授 稲垣 暢也, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Detrimental immune and inflammatory responses contribute to the pathogenesis of various conditions, including Crohn’s disease, Lupus, and sepsis.1,2,3 Unfortunately, novel treatments for detrimental immune and inflammatory responses have been met with little success. Mesenchymal stromal cells (MSCs) represent a promising cellular therapy to treat immune and inflammatory disorders due to their ability to suppress the immune system. However, despite their promise, clinical trials that have employed MSC cellular therapies have produced varying and sometimes conflicting results. These discrepancies have been partially attributed to the cellular heterogeneity within MSC populations. To address these discrepancies, I performed transcriptomic and proteomic analysis of MSCs with varying immunomodulatory capacity to identify robust immunomodulatory biomarkers and gain better mechanistic insights into MSC immunomodulatory function. In this study, MSCs with differing immunomodulatory function were identified and the effect of in vitro passaging and proinflammatory induction on immunomodulatory ability was characterized. To characterize MSC immunomodulatory control mechanisms, RNA sequencing and proteomic analyses were performed on MSCs with different immunomodulatory capabilities. These analyses enabled the identification of potential immunomodulatory biomarkers and regulatory mechanisms. Finally, to test the therapeutic efficacy of immunomodulatory MSC subpopulations, I developed a humanize mouse model for sepsis. Overall, this work contributes to our understanding of MSC immunomodulation and to the development of a robust MSC cellular therapeutics.

In the United States, nearly half a million bone graft operations are performed annually to repair defects arising from birth defects, trauma, and disease, making bone the second most transplanted tissue. Autogenous bone is the current gold standard for bone grafts; however it is in limited supply and results in a second injury at the donor site. A promising alternative is a tissue engineered bone graft composed of a biomaterial scaffold, pharmaceutics, and osteoprogenitor cells. One source of osteoprogenitor cells is bone marrow stroma, which can be obtained from the patient - minimizing the risk of an immune response - directed in vitro to proliferate, and differentiate into a bone-like tissue. To date, tissue engineered bone grafts have not been clinically effective; thus, strategies must be developed to improve efficacy. I hypothesize that to facilitate tissue healing in a manner similar to autogenous bone tissue engineering bone must possess a mineralized collagen matrix to support tissue integration, and angiogenic factors to stimulate vascular infiltration, and osteogenic factors to direct normal bone remodeling. I propose that these factors can be synthesized by osteoprogenitor cells in vitro when cultured under the appropriate conditions. Previous work has demonstrated that perfusion culture of osteoprogenitor cells within 3D scaffolds stimulates phenotypic markers of osteoblastic differentiation, but those studies did not determine whether the effects were a consequence of shear stress or increased nutrient availability. Consequently, this work has involved studies in a planar geometry, where nutrient effects are negligible. Three studies that characterize the effect of fluid flow on osteoblastic differentiation of osteoprogenitor cells are presented here. The objective of the first study was to determine the effect of shear stress magnitude on cell density and osteocalcin deposition. In this study, radial flow chambers were used to generate a spatially dependent range of shear stresses (0.36 to 2.7 dynes/cm2) across single substrates, and immunofluorescent techniques were used to assay cell phenotype as a function of shear stress. The objective of the second study was to determine the effect of the duration of fluid flow on cell density and phenotypic markers of differentiation. Here, parallel plate flow chambers were used to generate a single shear stress at the cell surface, and entire cell layers were assayed for expression of osteoblastic genes. The objective of the third study was to compare continuous and intermittent fluid flow strategies. In this study, a microprocessor-controlled actuator was added to the flow loop to periodically halt flow, and markers of mechanosensation and osteoblastic differentiation were measured. These studies demonstrated that shear stresses of 0.36 to 2.7 dynes/cm2 stimulate late phenotypic markers of osteoblastic differentiation but not cell proliferation. In addition, this osteogenic effect is sensitive to duration of fluid flow but insensitive to the magnitude of shear stress. Further, intermittent fluid flow enhances cell retention, biochemical markers of mechanotransduction, and synthesis of the angiogenic factor vascular endothelial growth factor (VEGF). Thus, these studies suggest that intermittent fluid flow may be an attractive component of a biodynamic bioreactor for in vitro manufacture of clinically effective tissue engineered bone grafts. Future studies will further investigate intermittent fluid flow strategies and three-dimensional studies with scaffolds suitable for bone tissue engineering. / Ph. D.

Bone is the second most transplanted tissue after blood and the need for bone graft materials continues to rise at an average annual growth rate of over 18%. An engineered bone substitute consisting of a bone-like extracellular matrix deposited on the internal pores of a resorbable biomaterial scaffold is postulated to stimulate normal bone remodeling when implanted in vivo. Part one of this engineering strategy, the deposition of bone-like extracellular matrix, can be achieved by the directed differentiation of progenitor cells such as bone marrow stromal cells (BMSCs). Part two of the engineering strategy, the biomaterial scaffold, can be fabricated with the appropriate mechanical properties using a synthetic polymer system with tunable properties like polyurethanes. Finally, BMSCs seeded within the biomaterial scaffold can be cultured in a perfusion flow bioreactor to stimulate osteoblastic differentiation and the deposition of bioactive factors. Using the three-part engineering strategy described, I hypothesize that the extracellular matrix produced by BMSCs can be modulated by two stimuli: the stiffness of the scaffold and perfusion flow. First, I propose that culturing BMSCs on polyurethane scaffolds with increasing stiffness will increase markers of osteoblastic differentiation. Secondly, I suggest that mechanically stimulating BMSCs with novel perfusion strategies will also increase markers of osteoblastic differentiation. In aim 1, a family of segmented degradable poly(esterurethane urea)s (PEUURs) were synthesized. The modulus of the PEUUR materials was systematically increased from 0.18 to 0.80 MPa by systematically increasing the molecular weight of the poly(ε-caprolactone) (PCL) soft segment from 1425 to 2700 Da. BMSCs were cultured on both rigid polymer films and on porous foam scaffolds to dissociate the effect of variation in polymer chemistry from the effect of scaffold modulus on cell phenotype. These studies demonstrated changes in osteoblastic differentiation as measured by prostaglandin E2 production, alkaline phosphatase activity (ALP) activity, and osteopontin gene expression. However, the increased levels of these phenotypic markers on the PCL 2700 material could not be attributed to scaffold chemistry or modulus. Instead, the differences may be related to polymer crystallinity or surface topography. In aim 2, novel dynamic perfusion strategies were used to investigate the influence of frequency on osteoblastic differentiation. BMSCs were seeded on porous foam scaffolds and exposed to both steady perfusion and pulsatile perfusion at 0.017, 0.050, and 0.083 Hz frequencies. The data presented here demonstrated that while some markers of osteoblastic phenotype such as ALP activity are enhanced by 0.05 Hz pulsatile flow over continuous flow, they are insensitive to frequency at low frequencies. Therefore, future studies will continue to investigate the effect of a larger range of frequencies. Additionally, fluid flow has also been shown to stimulate the deposition of bioactive factors such as BMP-2 and VEGF-A, and these growth factors are known to significantly enhance healing in bone defect models. Therefore, we plan to investigate the effect of dynamic flow strategies on the deposition of these bioactive factors. We propose that an engineered bone graft material containing a bone-like extracellular matrix and producing these growth factors will show more rapid formation of bone when implanted in vivo. / Ph. D.

Falls among the elderly are a major source of injury, often leading to serious fractures, hospitalization, and death. Osteoporosis (OP) is a global problem intimately related with these fractures, characterized by reduced bone mass, increased bone fragility. There exists a high failure rate in the translation of treatments to osteoporotic populations. Mesenchymal stromal cell (MSC) transplantation as a therapeutic strategy for OP has not yet been examined in clinical trials. This may be attributed to the mixed findings of pre-clinical studies aimed at determining the efficacy of MSC therapy towards bone regeneration in OP. The most common animal model of OP is ovariectomy (OVX) that simulates post-menopausal estrogen loss. A plethora of bone regeneration studies have used OVX models with 12-16 weeks post-OVX periods and have generally reported positive results from a variety of treatment modalities, including MSC therapy. However, the use of the minimum post-OVX period may not be appropriate to reflect the global changes in regenerative potential of OP patients. In our research group's previous study, MSC were isolated from a minimum 60 week post-OVX rat model, representing a severe case of OP. The MSC isolated from these animals are a unique cell population that we expect may better represent the outcomes of autologous cell therapies for the older patient population in the clinic. In the present study, adipose and bone marrow derived MSC from OVX and age-matched animals were evaluated for their osteogenic and adipogenic differentiation potentials in culture through passage 10. Results from this study suggest that bone marrow derived-MSC maintain their phenotype and functionality more effectively than adipose derived-MSC in OP. Further investigations used regenerative medicine approaches for cell expansion on keratin protein coated microcarriers in static culture. Hair-derived keratin biomaterials have demonstrated their utility as carriers of biologics and drugs for tissue engineering. An optimal microcarrier was selected that demonstrated superior retention of the protein coating through electrostatic interactions and high cell viability. Finally, the integration of cell-microcarriers into a perfusion bioreactor system was explored. Preliminary results demonstrated the feasibility of MSC growth and differentiation on microcarrier based packed beds. Moreover, AD-MSC from OP rats were unresponsive to both inductive media and shear stress related osteogenic cues. These results highlight the complexity and challenges associated with the MSC regenerative strategy. / Doctor of Philosophy / Osteoporosis is a skeletal disease that results in reduced bone mass, increased bone fragility and fracture risk. Osteoporotic patients who experience falls suffer serious fractures, hospitalization, and poor bone healing. Several different therapies have been developed for the treatment of osteoporosis, though many are unable to translate from the bench to the clinical population. A popular treatment being investigated is the application of mesenchymal stromal cells (MSC) for fracture repair and the reversal of osteoporotic bone losses. However, cells isolated from aged and osteoporotic patients have been shown to have deficient bone forming properties. Nevertheless, animal models of osteoporosis applying this treatment report amelioration of bone loss. This work seeks to examine a more clinically relevant rat model of osteoporosis. Typical osteoporosis models use an ovariectomy procedure to simulate post-menopausal bone loss on relatively young animals and conduct short-term studies. These studies may not accurately reflect the global regenerative changes in osteoporosis patients or the impaired MSC properties. Adipose and bone marrow derived MSC from a long term ovariectomy model were investigated for their regenerative potentials. MSC growth and bone forming potential was evaluated on keratin protein coated microcarriers in both static and perfusion cultures. Results from this study suggest that bone marrow derived MSC maintain their phenotype and functionality more effectively than adipose derived MSC in osteoporosis. Further preliminary results demonstrated the feasibility of MSC growth and differentiation on microcarrier based packed beds. These results highlight the complexity and challenges associated with the MSC regenerative strategy.

Células reticulares fibroblásticas (FRCs, gp38+ e CD31-) e células duplo negativas (DNCs, gp38- e CD31-) são células estromais encontradas em órgãos linfoides secundários, como linfonodos. Enquanto as FRCs têm sido amplamente estudadas, pouco se sabe ainda sobre DNCs. Apesar da função estrutural das FRCs nos linfonodos já estar bem estabelecida, estudos recentes indicam que as FRCs também desempenham um papel fundamental em processos imunológicos, por exemplo, migração celular, ativação e qualidade da resposta imune, além da participação na tolerância periférica. Outra célula estromal em constante estudo são as células-tronco mesenquimais (CTMs), principalmente encontradas na medula óssea. Estas células compartilham similaridades, como por exemplo; são células estromais encontradas em órgãos linfoides, apresentam morfologia e características semelhantes quando cultivadas in vitro e estão envolvidas na resposta imune por mecanismos semelhantes. As CTMs são provenientes de um órgão linfoide primário, cuja função principal não está relacionada à resposta imunológica, entretanto, de acordo com inúmeros trabalhos, estas células possuem capacidade de interferir na ativação de várias células do sistema imunológico. Portanto, nossa hipótese é de que as FRCs e DNCs, que se encontram em um órgão linfoide secundário, cuja função principal remete a resposta imunológica, apresentem também um papel regulador, descrito na literatura como tolerância periférica e contração de uma resposta imunológica já estabelecida. Em nosso estudo mostramos que FRCs e DNCs foram isoladas a partir de linfonodos humanos e devidamente caraterizadas. Evidenciamos que FRCs e DNCs atendem todos os critérios mínimos propostos pela sociedade internacional de terapia celular para serem consideradas células-tronco estromais. Além disso, mostramos que FRCs e DNCs influênciam a proliferação e a expressão de moléculas de homing em linfócitos alogênicos in vitro. Portanto, contribuimos de forma inédita para o entendimento funcional das FRCs e DNCs, visto que estudos em humanos envolvendo estas células são escassos / Fibroblastic reticular cells (FRCs, gp38+ e CD31-) and double-negative cells (DNCs, gp38- e CD31-) are stromal cells found in secondary lymphoid organs, such as lymph nodes. While the FRCs has been widely studied, little is known about DNCs. Despite the structural function of FRCs on lymph nodes is well established, recent studies indicate that FRCs also play a key role in immunological processes, for example, cell migration, immune response activation and quality, beyond their involvement in peripheral tolerance. Another stromal cell type in constant study are mesenchymal stem cells (MSCs), mainly found in bone marrow. These cells share similarities with FRCs and DNCs, for example; they are estromal cells found in lymphoid organs, they present similar morphology and characteristics when cultured in vitro and they are involved in the immune response by similar mechanisms. MSCs are derived from a primary lymphoid organ which the major function is not related to immune response, but according to numerous studies these cells have the capacity of the interfere on activation of various immune cells. Consequently, our hypothesis is that FRCs and DNCs, usually found in secondary lymphoid organ, display immune regulatory roles, which were described in the literature as peripheral tolerance and immune response contraction. In our study we showed that FRCs and DNCs were isolated from human lymph nodes and adequately characterized. We evidenced that FRCs and DNCs meet all minimum criteria proposed by the International Society of Cell Therapy to be considerate a stromal stem cell. Therefore, we contributed in an unpublished manner to the functional understanding of FRCs and DNCs, since human studies involving these cells are scarce

Células reticulares fibroblásticas (FRCs, gp38+ e CD31-) e células duplo negativas (DNCs, gp38- e CD31-) são células estromais encontradas em órgãos linfoides secundários, como linfonodos. Enquanto as FRCs têm sido amplamente estudadas, pouco se sabe ainda sobre DNCs. Apesar da função estrutural das FRCs nos linfonodos já estar bem estabelecida, estudos recentes indicam que as FRCs também desempenham um papel fundamental em processos imunológicos, por exemplo, migração celular, ativação e qualidade da resposta imune, além da participação na tolerância periférica. Outra célula estromal em constante estudo são as células-tronco mesenquimais (CTMs), principalmente encontradas na medula óssea. Estas células compartilham similaridades, como por exemplo; são células estromais encontradas em órgãos linfoides, apresentam morfologia e características semelhantes quando cultivadas in vitro e estão envolvidas na resposta imune por mecanismos semelhantes. As CTMs são provenientes de um órgão linfoide primário, cuja função principal não está relacionada à resposta imunológica, entretanto, de acordo com inúmeros trabalhos, estas células possuem capacidade de interferir na ativação de várias células do sistema imunológico. Portanto, nossa hipótese é de que as FRCs e DNCs, que se encontram em um órgão linfoide secundário, cuja função principal remete a resposta imunológica, apresentem também um papel regulador, descrito na literatura como tolerância periférica e contração de uma resposta imunológica já estabelecida. Em nosso estudo mostramos que FRCs e DNCs foram isoladas a partir de linfonodos humanos e devidamente caraterizadas. Evidenciamos que FRCs e DNCs atendem todos os critérios mínimos propostos pela sociedade internacional de terapia celular para serem consideradas células-tronco estromais. Além disso, mostramos que FRCs e DNCs influênciam a proliferação e a expressão de moléculas de homing em linfócitos alogênicos in vitro. Portanto, contribuimos de forma inédita para o entendimento funcional das FRCs e DNCs, visto que estudos em humanos envolvendo estas células são escassos / Fibroblastic reticular cells (FRCs, gp38+ e CD31-) and double-negative cells (DNCs, gp38- e CD31-) are stromal cells found in secondary lymphoid organs, such as lymph nodes. While the FRCs has been widely studied, little is known about DNCs. Despite the structural function of FRCs on lymph nodes is well established, recent studies indicate that FRCs also play a key role in immunological processes, for example, cell migration, immune response activation and quality, beyond their involvement in peripheral tolerance. Another stromal cell type in constant study are mesenchymal stem cells (MSCs), mainly found in bone marrow. These cells share similarities with FRCs and DNCs, for example; they are estromal cells found in lymphoid organs, they present similar morphology and characteristics when cultured in vitro and they are involved in the immune response by similar mechanisms. MSCs are derived from a primary lymphoid organ which the major function is not related to immune response, but according to numerous studies these cells have the capacity of the interfere on activation of various immune cells. Consequently, our hypothesis is that FRCs and DNCs, usually found in secondary lymphoid organ, display immune regulatory roles, which were described in the literature as peripheral tolerance and immune response contraction. In our study we showed that FRCs and DNCs were isolated from human lymph nodes and adequately characterized. We evidenced that FRCs and DNCs meet all minimum criteria proposed by the International Society of Cell Therapy to be considerate a stromal stem cell. Therefore, we contributed in an unpublished manner to the functional understanding of FRCs and DNCs, since human studies involving these cells are scarce

Master of Science / Department of Anatomy and Physiology / Mark L. Weiss / Wharton's jelly is a non-controversial source of mesenchymal stromal cells. Isolation of the cells is non-invasive and painless. The cells have been shown to have a wide array of therapeutic applications. They have improved symptoms when transplanted in a variety of animal disease models, can be used in tissue engineering applications to grow living tissue ex vivo for transplantation, and can be used as drug delivery vehicles in cancer therapy. The cells have also been shown to be non-immunogenic and immune suppressive. This thesis focuses on optimizing isolation protocols, culture protocols, cryopreservation, and characterization of cells in different growth conditions. Results from the experiments indicate that isolation of cells by enzyme digestion yields cells consistently, a freezing mixture containing 90% FBS and 10% DMSO confers maximum viability, and the expression of mesenchymal stromal cell consensus markers does not change with passage and cryopreservation. The results of the experiments also show that cells grow at a higher rate in 5% oxygen culture conditions compared to 21% oxygen culture conditions, serum does not have an effect on growth of the cells, serum and oxygen do not have effects on the expression of mesenchymal stromal cell consensus markers and the cells are stable without nuclear abnormalities when grown in 5% oxygen and serum free conditions for six passages after first establishing in serum conditions.

1. Einleitung Multipotente mesenchymale Stromazellen (MSC) stellen eine interessante Therapieoption in der regenerativen Medizin verschiedener Erkrankungen dar. Aufgrund ihrer Herkunft aus mesodermalem Gewebe ist ihr Einsatz in der Therapie von Sehnenerkrankungen als günstig anzusehen, wo sie bei Pferden bereits erfolgreich verwendet werden. Da dieser Erkrankungskomplex mit degenerativen Veränderungen der Achillessehne des Menschen vergleichbar ist, wäre eine Translation der gewonnenen Ergebnisse in die Humanmedizin wünschenswert. Die zugrunde liegenden Wirkmechanismen bei der Sehnenregeneration sind allerdings bis zum heutigen Tage noch nicht vollständig geklärt. Unter anderem wird eine tenogene Differenzierung der MSC mit nachfolgender Produktion von extrazellulärer Matrix (EZM) diskutiert. Als Nachweis hierfür wird die Genexpression von Matrixproteinen sowie Transkriptionsfaktoren angesehen. Die Isolation von MSC ist aus verschiedenen Geweben möglich; allerdings haben Untersuchungen deutliche Unterschiede in den in-vitro-Charakteristika zwischen den Zellquellen aufgezeigt. Trotz dieser unterschiedlichen Eigenschaften fasst die International Society for Cellular Therapy (ISCT) seit 2006 humane MSC als plastikadhärente Zellen mit tripotentem Differenzierungspotential sowie einem definierten Antigenprofil zusammen. Um eine Vergleichbarkeit equiner und humaner MSC und somit eine bessere Übertragbarkeit gewonnener Erkenntnisse aus der Pferdemedizin zu erreichen, steht aktuell die Untersuchung der geforderten Antigenexpression noch aus. 2. Ziele der Untersuchung In der vorliegenden Arbeit sollte daher erstmalig eine vollständige Charakterisierung des geforderten Antigenprofils equiner MSC aus fünf verschiedenen Quellen durchgeführt werden, um einen Vergleich mit humanen Zellen zu ermöglichen. Zudem sollte eine vergleichende Darstellung der Sehnenmarkerexpression durchgeführt werden, welche das Wissen um die in-vitro-Eigenschaften von MSC erweitern und in Folge zur Auswahl einer optimal für die Therapie von Sehnenerkrankungen geeigneten Zellquelle beitragen soll. 3. Materialien und Methoden In der ersten Studie wurden equine MSC aus Knochenmark, Fettgewebe, Nabelschnurblut, Nabelschnurgewebe und Sehnengewebe bis zur Passage 3 kultiviert und anschließend mittels Durchflusszytometrie auf das Vorkommen der Antigene CD 29, CD 44, CD 73, CD 90 und CD 105 sowie das Fehlen der Antigene CD 14, CD 34, CD 45, CD 79α und MHC II untersucht. In der zweiten Studie wurde eine Genexpressionsanalyse der Sehnenmarker Kollagen 1A2, Kollagen 3A1, Decorin, Tenascin-C und Skleraxis vergleichend mittels Echtzeitpolymerasekettenreaktion an den isolierten Zellen durchgeführt. In beiden Studien wurde eine Probenzahl von n= 6 für jede Zellquelle untersucht. 4. Ergebnisse Keine der untersuchten Zellquellen erfüllte die MSC-Definition der ISCT bezüglich des Antigenprofils. Insbesondere durch den fehlenden Nachweis CD 73 (< 3,07 %) in allen untersuchten Proben unterscheiden sich equine und humane MSC. Die einzigen stabil exprimierten Antigene sind die zusätzlich untersuchten Proteine CD 29 (37,5 % - 65,42 %) und CD 44 (32,2 % - 97,18 %). Das Vorkommen CD 105 konnte in MSC aus Fett- und Sehnengewebe belegt werden. Zusätzlich war ein Nachweis von CD 90 in MSC aus Fettgewebe möglich, welche somit die größte Ähnlichkeit mit der humanen Zellpopulation aufweisen. Die Studie zur Genexpressionsanalyse weist auf eine Basisexpression von Kollagen 1A2, 3A1 und Decorin in MSC aus verschiedenen Quellen hin, welche über der von nativem Sehnengewebe liegt. Auch hier weisen wiederum MSC aus Fettgewebe die höchste Expression auf. 5. Schlussfolgerungen Die vorliegende Arbeit leistet einen Beitrag zu einer vertiefenden in-vitroCharakterisierung equiner MSC. Das Antigenprofil equiner MSC ist nicht vollständig mit dem humaner identisch. Eine abschließende Beurteilung sollte durch Untersuchungen mit spezies-spezifischen Antikörpern erfolgen. Die Ergebnisse der Genexpressionsanalyse unterstützen die Theorie, dass MSC die Sehnenheilung durch Produktion von extrazellulärer Matrix beeinflussen. Der Einsatz von MSC aus Fettgewebe in der Therapie von Sehnenerkrankungen sollte forciert werden, da ihre hohe Sehnenmarkerexpression einen Hinweis auf eine Verbesserung der Sehnenregeneration darstellt.

The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversial. We therefore analyzed the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Furthermore, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha in patients’ cells culture supernatants were almost 2-fold lower (P<0.01) than those in controls and this was paralleled by a reduced induction of migration of CD34+ hematopoietic cells. Co-cultures of mesenchymal stromal cells from patients with CD34+ cells from healthy donors resulted in reduced numbers of cobblestone area-forming cells and fewer colony-forming units. Exposure of stromal cells from patients and controls to lenalidomide led to a further reduction of stromal derived factor 1-alpha secretion and cobblestone area formation, respectively. Moreover, lenalidomide pretreatment of mesenchymal stromal cells from patients with low but not high-risk myelodysplastic syndrome was able to rescue impaired erythroid and myeloid colony formation of early hematopoietic progenitors. In conclusion, our analyses support the notion that the stromal microenvironment is involved in the pathophysiology of myelodysplastic syndrome thus representing a potential target for therapeutic interventions.