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Papers by Misty Marchioni

Stem Cells International, Nov 25, 2019

Stem cells can be isolated from various human tissues including bone marrow (BM) and adipose tiss... more Stem cells can be isolated from various human tissues including bone marrow (BM) and adipose tissue (AT). Our study outlines a process to isolate adult stem cells from deceased donors. We have shown that cell counts obtained from deceased donor BM were within established living donor parameters. Evaluation of demographic information exhibited a higher percentage of hematopoietic stem cells (HSC) in males versus females, as well as a higher percentage of HSC in the age bracket of 25 years and under. For the first time, we show that deceased donor femur BM grew cell colonies. Our introduction of new technology for nonenzymatic AT processing significantly increased cell recovery over the traditional enzymatic processing method. Cell counts from the deceased donor AT exceeded living donor parameters. Furthermore, our data illustrated that AT from female donors yielded a much higher number of total nucleated cells (TNC) than males. Together, our data demonstrates that our approach to isolate stem cells from deceased donors could be a routine practice to provide a viable alternative to living donor stem cells. This will offer increased accessibility for patients awaiting stem cell therapies.

Clinical & Translational Immunology

Objectives. There are four immunoglobulin (IgG) subtypes that have varying complement-activating ... more Objectives. There are four immunoglobulin (IgG) subtypes that have varying complement-activating ability: strong (IgG3 and IgG1) and weak (IgG2 and IgG4). The standard flow cytometric crossmatch (FCM) assay does not distinguish between the various subtypes of the IgG molecule. This study outlines the development and use of a novel cell-based IgG subtype-specific FCM assay that is able to detect the presence of and quantitate the IgG subtypes bound to donor cells. Methods. A six-colour lyophilised reagent was designed that specifically detects the four IgG subtypes, as well as distinguishes between T cells and B cells in the lymphocyte population. To test the efficacy of this reagent, a retrospective evaluation of a group of highly sensitised patients awaiting heart and kidney transplant was carried out, who, because of positive standard FCM results, had been deemed incompatible with numerous prior potential donors. Results. Observations in this study demonstrate that the positive standard FCM results were mainly because of the presence of noncomplement-activating IgG2 or IgG4 antibodies. The results were supported by the absence of C3d-binding donor-specific antibodies (DSA) and a negative complement-dependent cytotoxicity crossmatch (CDC). Conclusion. Preliminary data presented in this study demonstrate the reliability of the novel IgG subtype assay to detect the presence of pretransplant, complement-activating antibodies bound to donor cells. The knowledge gained from the IgG subtype assay and the C3d-binding specificities of DSAs provides improved identification of donor suitability in pretransplant patients, potentially increasing the number of transplants.

Pharmaceutics, Apr 4, 2022

Journal of Stem Cells and Regenerative Medicine, 2020

There is an emerging need for the rapid generation of functional beta cells that can be used in c... more There is an emerging need for the rapid generation of functional beta cells that can be used in cell replacement therapy for the treatment of type 1 diabetes (T1D). Differentiation of stem cells into insulin-producing cells provides a promising strategy to restore pancreatic endocrine function. Stem cells can be isolated from various human tissues including adipose tissue (AT). Our study outlines a novel, non-enzymatic process to harvest mesenchymal stem cells (MSC) from research-consented, deceased donor AT. Following their expansion, MSC were characterised morphologically and phenotypically by flow cytometry to establish their use for downstream differentiation studies. MSC were induced to differentiate into insulin-producing beta cells using a step-wise differentiation medium. The differentiation was evaluated by analysing the morphology, dithizone staining, immunocytochemistry, and expression of pancreatic beta cell marker genes. We stimulated the beta cells with different concentrations of glucose and observed a dose-dependent increase in gene expression. In addition, an increase in insulin and c-Peptide secretion as a function of glucose challenge confirmed the functionality of the differentiated beta cells. The differentiation of adipose-derived MSC into beta cells has been well established. However, our data demonstrates, for the first time, that the ready availability and properties of MSC isolated from deceased donor adipose tissue render them well-suited as a source for increased production of functional beta cells. Consequently, these cells can be a promising therapeutic approach for cell replacement therapy to treat patients with T1D.

Human Immunology, 2015

The new Kidney Allocation System (KAS) implemented on December 4, 2014 was developed to improve t... more The new Kidney Allocation System (KAS) implemented on December 4, 2014 was developed to improve the discard rates of kidneys, give patients with extensive wait time and high Panel Reactive Antibody (PRA) access to transplantation and improve re-transplant rates. Under the new system, highly sensitized patients are given priority by allowing additional points for candidates with Calculated Panel Reactive Antibody (CPRA) >98% thus enabling them to receive regional and national priority kidney offers. Methods: The NJ Sharing Network (NJSN) is an organ procurement organization equipped with a transplant laboratory. This study evaluated the impact of the new KAS on NJSN's transplant laboratory operations by measuring the number of (1) deceased donor (DD) crossmatches, (2) recipients transplanted, (3) blood samples shipped after the new KAS was implemented, and (4) kidneys discarded, and compared them to the same parameters prior to the KAS implementation. Results: More crossmatches were performed on import DD than local DD. The percentage of imported DD that were crossmatched with recipients was 58% between December 4, 2014 and April 2, 2015 as compared to 22% during the same timeframe the prior year. The number of virtual crossmatches performed increased significantly after implementation of the new KAS. There were 21 patients with CPRAs over 98% who received a kidney transplant between December 19, 2014 and May 1, 2015. Of the 21 transplanted organs, only 4 were from local DD, the remaining were import donors. There were 0 transplants from patients with CPRAs over 98% from the same timeframe the prior year. There were 22 DD kidney blood samples shipped to other transplant laboratories during the first quarter of 2015 as compared to 4 blood samples shipped during the same timeframe in 2014 resulting in a 5.5 fold increase. The 2015 year-to-date kidney discard rate is 24% as compared to 17% in 2014, and 10% in 2013. Conclusions: We observed (1) an increase of imported DD being crossmatched, (2) patients with CPRAs over 98% receiving transplants, (3) an increase in the number of blood samples being shipped to other transplant laboratories and (4) an increase in the number of kidneys being discarded.

Ash Annual Meeting Abstracts, Nov 16, 2004

Controlled rate freezing is recognized as a method of choice for the cryopreservation of blood ce... more Controlled rate freezing is recognized as a method of choice for the cryopreservation of blood cells. DMSO, the cryoprotectant most commonly used for cryopreservation of cells, is well known to affect cell viability. It is therefore imperative to insure that cells are frozen without any appreciable delay after the addition of DMSO. Addition of cryoprotectant may take between 5–10 minutes per processed cord blood unit. In situations involving large scale processing, such as 5–10 cord blood units simultaneously, delay will occur, which could cause adverse affects on cell viability when controlled rate freezing is utilized This study was designed to compare the effect of controlled with two non-controlled rate freezing methods on cord blood stem cell viability. After adding DMSO to final concentration of 10%, each cord blood unit was split into two 25 ml volumes and enclosed in metal freezing canisters. The UCB units were covered by styrofoam sleeves and then placed directly into either a −80°C or a −140°C mechanical freezer. A corresponding portion of each unit was frozen utilizing a Forma Scientific, model 1010 controlled rate freezer. Approximately 30 days after, matching units were thawed in a 37°C water bath and tested without washing. Total Nucleated Cell count, Total CD34 count, 7AAD viability and colony forming units assay were performed on fresh and cryopreserved/thawed samples. Statistical pair analysis using student-t test was performed to indicate any statistically significant differences between each procedures. (table1) (table2) Results indicate that all three methods produces similar cell viability as measured by total nucleated cell recovery, total CD34 counts, 7AAD viability and colony forming assays. Analysis of fresh and frozen samples did not indicate that controlled rate freezing produces a better quality product after thawing compared to two uncontrolled freezing methods. Controlled rate freezing vs −80oC dump method° N=5 Fresh Sample Controlled Rate Freezing Non controlled Freezing at −80oC TNC (x10^6) 412.4 ±37.06 397.6±15.65 406.7±24.78 Total CD34+ (10^6) 0.23±0.08 0.19±0.08 p<0.05 0.19±0.07 p<0.05 Viability (%) 97.5±1.67 94.8±2.28 p<0.05 93.4±3.51 p<0.05 Total # of Colonies (x10^6) 0.27±0.09 0.21±0.10 p<0.05 0.20±0.08 p<0.05 Controlled rate freezing vs −140oC dump method N=5 Fresh Sample Controlled Rate Freezing Non Controlled −140oC TNC (10^6) 365.4±100.87 372.1±102.18 365.9±104.9 Total CD34+ (x10^6) 0.28±0.08 0.023±0.05 p<0.05 0.23±0.05 p<0.05 Viability (5) 96.0±3.94 96.2±2.28 p<0.05 96.2±1.79 Total # of Colonies (10^6) 0.27±0.13 0.27±0.11 0.24±0.15

Stem Cells International, Nov 25, 2019

Stem cells can be isolated from various human tissues including bone marrow (BM) and adipose tiss... more Stem cells can be isolated from various human tissues including bone marrow (BM) and adipose tissue (AT). Our study outlines a process to isolate adult stem cells from deceased donors. We have shown that cell counts obtained from deceased donor BM were within established living donor parameters. Evaluation of demographic information exhibited a higher percentage of hematopoietic stem cells (HSC) in males versus females, as well as a higher percentage of HSC in the age bracket of 25 years and under. For the first time, we show that deceased donor femur BM grew cell colonies. Our introduction of new technology for nonenzymatic AT processing significantly increased cell recovery over the traditional enzymatic processing method. Cell counts from the deceased donor AT exceeded living donor parameters. Furthermore, our data illustrated that AT from female donors yielded a much higher number of total nucleated cells (TNC) than males. Together, our data demonstrates that our approach to isolate stem cells from deceased donors could be a routine practice to provide a viable alternative to living donor stem cells. This will offer increased accessibility for patients awaiting stem cell therapies.

Clinical & Translational Immunology

Objectives. There are four immunoglobulin (IgG) subtypes that have varying complement-activating ... more Objectives. There are four immunoglobulin (IgG) subtypes that have varying complement-activating ability: strong (IgG3 and IgG1) and weak (IgG2 and IgG4). The standard flow cytometric crossmatch (FCM) assay does not distinguish between the various subtypes of the IgG molecule. This study outlines the development and use of a novel cell-based IgG subtype-specific FCM assay that is able to detect the presence of and quantitate the IgG subtypes bound to donor cells. Methods. A six-colour lyophilised reagent was designed that specifically detects the four IgG subtypes, as well as distinguishes between T cells and B cells in the lymphocyte population. To test the efficacy of this reagent, a retrospective evaluation of a group of highly sensitised patients awaiting heart and kidney transplant was carried out, who, because of positive standard FCM results, had been deemed incompatible with numerous prior potential donors. Results. Observations in this study demonstrate that the positive standard FCM results were mainly because of the presence of noncomplement-activating IgG2 or IgG4 antibodies. The results were supported by the absence of C3d-binding donor-specific antibodies (DSA) and a negative complement-dependent cytotoxicity crossmatch (CDC). Conclusion. Preliminary data presented in this study demonstrate the reliability of the novel IgG subtype assay to detect the presence of pretransplant, complement-activating antibodies bound to donor cells. The knowledge gained from the IgG subtype assay and the C3d-binding specificities of DSAs provides improved identification of donor suitability in pretransplant patients, potentially increasing the number of transplants.

Pharmaceutics, Apr 4, 2022

Journal of Stem Cells and Regenerative Medicine, 2020

There is an emerging need for the rapid generation of functional beta cells that can be used in c... more There is an emerging need for the rapid generation of functional beta cells that can be used in cell replacement therapy for the treatment of type 1 diabetes (T1D). Differentiation of stem cells into insulin-producing cells provides a promising strategy to restore pancreatic endocrine function. Stem cells can be isolated from various human tissues including adipose tissue (AT). Our study outlines a novel, non-enzymatic process to harvest mesenchymal stem cells (MSC) from research-consented, deceased donor AT. Following their expansion, MSC were characterised morphologically and phenotypically by flow cytometry to establish their use for downstream differentiation studies. MSC were induced to differentiate into insulin-producing beta cells using a step-wise differentiation medium. The differentiation was evaluated by analysing the morphology, dithizone staining, immunocytochemistry, and expression of pancreatic beta cell marker genes. We stimulated the beta cells with different concentrations of glucose and observed a dose-dependent increase in gene expression. In addition, an increase in insulin and c-Peptide secretion as a function of glucose challenge confirmed the functionality of the differentiated beta cells. The differentiation of adipose-derived MSC into beta cells has been well established. However, our data demonstrates, for the first time, that the ready availability and properties of MSC isolated from deceased donor adipose tissue render them well-suited as a source for increased production of functional beta cells. Consequently, these cells can be a promising therapeutic approach for cell replacement therapy to treat patients with T1D.

Human Immunology, 2015

The new Kidney Allocation System (KAS) implemented on December 4, 2014 was developed to improve t... more The new Kidney Allocation System (KAS) implemented on December 4, 2014 was developed to improve the discard rates of kidneys, give patients with extensive wait time and high Panel Reactive Antibody (PRA) access to transplantation and improve re-transplant rates. Under the new system, highly sensitized patients are given priority by allowing additional points for candidates with Calculated Panel Reactive Antibody (CPRA) >98% thus enabling them to receive regional and national priority kidney offers. Methods: The NJ Sharing Network (NJSN) is an organ procurement organization equipped with a transplant laboratory. This study evaluated the impact of the new KAS on NJSN's transplant laboratory operations by measuring the number of (1) deceased donor (DD) crossmatches, (2) recipients transplanted, (3) blood samples shipped after the new KAS was implemented, and (4) kidneys discarded, and compared them to the same parameters prior to the KAS implementation. Results: More crossmatches were performed on import DD than local DD. The percentage of imported DD that were crossmatched with recipients was 58% between December 4, 2014 and April 2, 2015 as compared to 22% during the same timeframe the prior year. The number of virtual crossmatches performed increased significantly after implementation of the new KAS. There were 21 patients with CPRAs over 98% who received a kidney transplant between December 19, 2014 and May 1, 2015. Of the 21 transplanted organs, only 4 were from local DD, the remaining were import donors. There were 0 transplants from patients with CPRAs over 98% from the same timeframe the prior year. There were 22 DD kidney blood samples shipped to other transplant laboratories during the first quarter of 2015 as compared to 4 blood samples shipped during the same timeframe in 2014 resulting in a 5.5 fold increase. The 2015 year-to-date kidney discard rate is 24% as compared to 17% in 2014, and 10% in 2013. Conclusions: We observed (1) an increase of imported DD being crossmatched, (2) patients with CPRAs over 98% receiving transplants, (3) an increase in the number of blood samples being shipped to other transplant laboratories and (4) an increase in the number of kidneys being discarded.

Ash Annual Meeting Abstracts, Nov 16, 2004

Controlled rate freezing is recognized as a method of choice for the cryopreservation of blood ce... more Controlled rate freezing is recognized as a method of choice for the cryopreservation of blood cells. DMSO, the cryoprotectant most commonly used for cryopreservation of cells, is well known to affect cell viability. It is therefore imperative to insure that cells are frozen without any appreciable delay after the addition of DMSO. Addition of cryoprotectant may take between 5–10 minutes per processed cord blood unit. In situations involving large scale processing, such as 5–10 cord blood units simultaneously, delay will occur, which could cause adverse affects on cell viability when controlled rate freezing is utilized This study was designed to compare the effect of controlled with two non-controlled rate freezing methods on cord blood stem cell viability. After adding DMSO to final concentration of 10%, each cord blood unit was split into two 25 ml volumes and enclosed in metal freezing canisters. The UCB units were covered by styrofoam sleeves and then placed directly into either a −80°C or a −140°C mechanical freezer. A corresponding portion of each unit was frozen utilizing a Forma Scientific, model 1010 controlled rate freezer. Approximately 30 days after, matching units were thawed in a 37°C water bath and tested without washing. Total Nucleated Cell count, Total CD34 count, 7AAD viability and colony forming units assay were performed on fresh and cryopreserved/thawed samples. Statistical pair analysis using student-t test was performed to indicate any statistically significant differences between each procedures. (table1) (table2) Results indicate that all three methods produces similar cell viability as measured by total nucleated cell recovery, total CD34 counts, 7AAD viability and colony forming assays. Analysis of fresh and frozen samples did not indicate that controlled rate freezing produces a better quality product after thawing compared to two uncontrolled freezing methods. Controlled rate freezing vs −80oC dump method° N=5 Fresh Sample Controlled Rate Freezing Non controlled Freezing at −80oC TNC (x10^6) 412.4 ±37.06 397.6±15.65 406.7±24.78 Total CD34+ (10^6) 0.23±0.08 0.19±0.08 p<0.05 0.19±0.07 p<0.05 Viability (%) 97.5±1.67 94.8±2.28 p<0.05 93.4±3.51 p<0.05 Total # of Colonies (x10^6) 0.27±0.09 0.21±0.10 p<0.05 0.20±0.08 p<0.05 Controlled rate freezing vs −140oC dump method N=5 Fresh Sample Controlled Rate Freezing Non Controlled −140oC TNC (10^6) 365.4±100.87 372.1±102.18 365.9±104.9 Total CD34+ (x10^6) 0.28±0.08 0.023±0.05 p<0.05 0.23±0.05 p<0.05 Viability (5) 96.0±3.94 96.2±2.28 p<0.05 96.2±1.79 Total # of Colonies (10^6) 0.27±0.13 0.27±0.11 0.24±0.15