Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) can be induced to express cardiac-specific markers by embryonic cardiomyocytes in vitro. To determine whether this phenomenon occurs in vivo, we have developed a cell transplantation system using zebrafish embryonic recipients. We were unable to isolate expandable zebrafish kidney stromal (ZKS) cells from the kidney, the human BM equivalent; hence, we analyzed the established ZKS1 cell line. We found that ZKS1 expresses stromal genes, but also expresses hematopoietic genes not normally expressed by MSCs. Furthermore, we were unable to differentiate ZKS1 cells into adipocytes, osteoblasts or cardiomyocytes in vitro. We created a transgenic ZKS1(CMV:eGFP) cell line which, after transplantation into zebrafish blastulae, was observed within the host heart, among other tissues. Finally, pT2/S2tnnt2-GM2 and pT2/S2tnnt2-DsRed transposons were generated to mark ZKS1 cardiac differentiation. The zebrafish model established here will be useful for studying the molecular mechanisms of exogenous MSC cardiac differentiation in vivo.

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) can be induced to express cardiac-specific markers by embryonic cardiomyocytes in vitro. To determine whether this phenomenon occurs in vivo, we have developed a cell transplantation system using zebrafish embryonic recipients. We were unable to isolate expandable zebrafish kidney stromal (ZKS) cells from the kidney, the human BM equivalent; hence, we analyzed the established ZKS1 cell line. We found that ZKS1 expresses stromal genes, but also expresses hematopoietic genes not normally expressed by MSCs. Furthermore, we were unable to differentiate ZKS1 cells into adipocytes, osteoblasts or cardiomyocytes in vitro. We created a transgenic ZKS1(CMV:eGFP) cell line which, after transplantation into zebrafish blastulae, was observed within the host heart, among other tissues. Finally, pT2/S2tnnt2-GM2 and pT2/S2tnnt2-DsRed transposons were generated to mark ZKS1 cardiac differentiation. The zebrafish model established here will be useful for studying the molecular mechanisms of exogenous MSC cardiac differentiation in vivo.

The studies in this thesis examined piglet gonocyte identification, isolation, purification, preservation and potential for initiation of spermatogenesis after transplantation into irradiated recipient testes. As a first step, we characterized a previously non-described auto-fluorescence in the piglet testis tissue. This auto-fluorescence mainly originated from granules among the testis interstitial cells, and we found that its interference with immuno-fluorescence can be overcome using Sudan black staining. We also showed that porcine gonocytes can be specifically labelled with the lectin Dolichos biflorus agglutinin (DBA). To optimize gonocyte isolation, we found that ~9-fold more live cells could be harvested by enzymatic digestion of testis tissues than with mechanical methods. However, the proportion of gonocytes (~7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. We then developed a novel three-step strategy for isolation of gonocytes by combining enzymatic digestion and vortexing, resulting in a gonocyte proportion of ~40% (~5-fold more than that from conventional methods). For short-term preservation of testis cells, we found that the survival of testis cells under hypothermic conditions was dependent on the cell type, and affected by storage duration, temperature and medium used. More than 80% of live testis cells survived the 6-day hypothermic preservation period in 20% FBS-L15, without visible changes to the cell culture potential or gonocyte proportion. In another experiment where testis tissues were maintained under hypothermic conditions, we found that ~25% of testis cells could survive for 6 days if preserved in HypoThermosol-FRS solution (HTS-FRS), without morphological changes. To purify gonocytes, we showed that centrifugation of testis cells using 17% Nycodenz can lead to precipitation of gonocytes in pellets (with a purity of > 80%). We also found that pre-coating tissue culture plates with both fibronectin and poly-D-lysine can result in the negative selection of gonocytes (with a purity of up to 85%). We subsequently showed that further purification of gonocytes (to > 90%) could be achieved by combining the two latter approaches. To prepare recipients for germ cell transplantation, we used local irradiation of piglet testes which reduced testis growth, decreased seminiferous tubule diameters and completely eliminated spermatogenesis at 4 months post-irradiation. Compared with the absence of endogenous spermatogenesis in the control testes, spermatogenesis up to elongating spermatids was observed in the irradiated testes after gonocyte transplantation. In summary, we investigated several critical elements in the study and manipulation of gonocytes in a large animal model.

The studies in this thesis examined piglet gonocyte identification, isolation, purification, preservation and potential for initiation of spermatogenesis after transplantation into irradiated recipient testes. As a first step, we characterized a previously non-described auto-fluorescence in the piglet testis tissue. This auto-fluorescence mainly originated from granules among the testis interstitial cells, and we found that its interference with immuno-fluorescence can be overcome using Sudan black staining. We also showed that porcine gonocytes can be specifically labelled with the lectin Dolichos biflorus agglutinin (DBA). To optimize gonocyte isolation, we found that ~9-fold more live cells could be harvested by enzymatic digestion of testis tissues than with mechanical methods. However, the proportion of gonocytes (~7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. We then developed a novel three-step strategy for isolation of gonocytes by combining enzymatic digestion and vortexing, resulting in a gonocyte proportion of ~40% (~5-fold more than that from conventional methods). For short-term preservation of testis cells, we found that the survival of testis cells under hypothermic conditions was dependent on the cell type, and affected by storage duration, temperature and medium used. More than 80% of live testis cells survived the 6-day hypothermic preservation period in 20% FBS-L15, without visible changes to the cell culture potential or gonocyte proportion. In another experiment where testis tissues were maintained under hypothermic conditions, we found that ~25% of testis cells could survive for 6 days if preserved in HypoThermosol-FRS solution (HTS-FRS), without morphological changes. To purify gonocytes, we showed that centrifugation of testis cells using 17% Nycodenz can lead to precipitation of gonocytes in pellets (with a purity of > 80%). We also found that pre-coating tissue culture plates with both fibronectin and poly-D-lysine can result in the negative selection of gonocytes (with a purity of up to 85%). We subsequently showed that further purification of gonocytes (to > 90%) could be achieved by combining the two latter approaches. To prepare recipients for germ cell transplantation, we used local irradiation of piglet testes which reduced testis growth, decreased seminiferous tubule diameters and completely eliminated spermatogenesis at 4 months post-irradiation. Compared with the absence of endogenous spermatogenesis in the control testes, spermatogenesis up to elongating spermatids was observed in the irradiated testes after gonocyte transplantation. In summary, we investigated several critical elements in the study and manipulation of gonocytes in a large animal model.

Syftet med den här undersökningen var att beskriva hur anhöriga upplever och hanterar sin livssituation, med fokus på familjen och deras egen hälsa, under den akuta fasen vid allogen stamcellstransplantation. Undersökningen ägde rum på Centrum för Allogen Stamcellstransplantation vid Karolinska Universitetssjukhuset Huddinge. Sju anhöriga till patienter som genomgick en allogen stamcellstransplantation deltog. Metoden som användes var att genom semistrukturerade intervjuer låta anhöriga själva berätta om sin livssituation. Dessa intervjuer analyserades sedan med hjälp av kvalitativ innehållsanalys vilket resulterade i att temat den nuvarande situationen formulerades. Temat bestod av tre kategorier livspussel, familj och hälsa och hade tio underkategorier totalt. Resultatet blev att när anhöriga får berätta om hur de upplever sin livssituation berättar de om hur de mobiliserar och fokuserar all sin kraft och alla sina resurser till den nuvarande situationen som de befinner sig i. Slutsatsen blev att vidare forskning för att utveckla interventioner för dessa anhöriga behövs, där sårbarhet upptäcks och inre och yttre resurser uppmärksammas och stärks. Att fokusera på anhöriga och den sjuka familjemedlemmen samtidigt, som en enhet, främjar hela familjens hälsa och medför därför en vinst, inte bara för de anhöriga utan även för patienten och dess tillfrisknande. / The aim of this study was to describe family members’ experience of life during the acute phase of allogenic stem cell transplantation. Seven family members of patients at, “Centrum for Allogenic Stem cell transplantation” Karolinska University Hospital in Huddinge participated. The method used was semistruktured interviews that allowed the respondents to tell about their living situation. These interviews were then analyzed through qualitative content analysis. The findings were a theme named; this present time consisting of three categories: living-pieces, family and health. There were also ten subcategories all together. The result was that when family members gives the opportunity to, with their own words, describe and tell about their living situation they tell about the present time and how they focus and mobilise all their strength and energy on their sick family member to get well. The conclusion of this study was that further research and development of interventions, for family members, are necessary and to focus on the family as a unite is an absolute condition in order to maintain health and prevent physical- and mental illness for the whole family. This ought to be beneficial not only for the family but also for the patients’ recovery.

Clinical trials investigating autologous stem cell transplantation (ASCT) have historically excluded elderly patients due to the risk of treatment-related morbidity related to the administration of high dose chemotherapy. While the availability of this procedure continues to expand, the elderly still represent a population for which the role of ASCT needs to be fully defined. 201 patients who underwent autologous stem cell transplantation (ASCT) for Non Hodgkins lymphoma (NHL) at a single institution following BEAM conditioning between January 1, 2000 and December 31, 2007 were retrospectively identified from the Yale University School of Medicine Bone Marrow Transplant Database. 67 patients were older than 60 years at the time of transplantation (median age 65, range 60 75) and were compared to a matched group of 134 patients transplanted during the same time period. These groups were extremely well-matched for all demographics such as gender, NHL histology, performance status, and comorbidities. Most patients had advanced stage disease at diagnosis and were transplanted at first or second remission. Diffuse large B-cell and mantle cell lymphoma were the most common subtypes but other subtypes were represented. The elderly group experienced significantly more serious toxicities within the first 100 days (63%) when compared to the control group (42%). However, there were no statistical differences (p<0.0001) between the groups regarding specific organ system toxicities. The 1-year non-relapse mortality (3%) was not significantly different when compared to the younger cohort (1%). At a median follow-up of 31 months the median overall survival is 85 months in the elderly group and at a median follow up of 33 months in the younger group the median overall survival has not yet been reached. The overall survival at 3 years is 74% and 75% respectively (p=0.91). The disease-free survival at 3 years is 48% in the elderly group compared to 58% in the control group (p=0.66). By univariate analysis, age >60 years (RR 3.1, 95% CI 1.7 5.7, p=0.004) was the only factor predictive of developing a serious toxicity from ASCT within the first 100 days. HCT-CI score (RR 2, 95% CI 1 4, p=0.043) was the only factor associated with significantly worse overall survival. Autologous stem cell transplantation can be safely performed in selected patients older than 60 years with chemosensitive NHL. Although elderly patients appear more likely to develop acute toxicities, the outcomes are similar to that of younger patients with respect to non-relapse mortality, disease-free survival, and overall survival.

Transplantation of olfactory ensheathing cells (OECs) into injured spinal cord results in improved functional outcome through axonal regeneration, remyelination, and neuroprotection. However, because little is known of the fate of OECs transplanted into injured peripheral nerve, their myelin forming potential requires investigation. To study these issues OECs were isolated from the olfactory bulbs of adult green fluorescent protein (GFP)-expressing transgenic rats and transplanted into a sciatic nerve crush lesions. Five weeks to six months after transplantation the nerves were studied histologically and it was determined that GFP-expressing OECs survived in the lesion and distributed longitudinally across the lesion zone. Immunostaining revealed a high density of isoform Nav1.6 at the newly formed nodes of Ranvier which were flanked by paranodal Caspr staining. Immuno-electron microscopy for GFP revealed transplanted OECs form peripheral type myelin. These results indicate that transplanted OECs extensively integrate into transected peripheral nerve, form myelin on regenerated peripheral nerve fibers, and reconstruct nodes of Ranvier with proper sodium channel structure.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 85-105).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 101-113).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010. / Härtill 5 uppsatser.