Bioluminescence Imaging and ICP-MS Associated with SPION as a Tool for Hematopoietic Stem and Progenitor Cells Homing and Engraftment Evaluation.

Bone marrow transplantation is a treatment for a variety of hematological and non-hematological diseases. For the transplant success, it is mandatory to have a thriving engraftment of transplanted cells, which directly depends on their homing. The present study proposes an alternative method to evaluate the homing and engraftment of hematopoietic stem cells using bioluminescence imaging and inductively coupled plasma mass spectrometry (ICP-MS) associated with superparamagnetic iron oxide nanoparticles. We have identified an enriched population of hematopoietic stem cells in the bone marrow following the administration of Fluorouracil (5-FU). Lately, the cell labeling with nanoparticles displayed the greatest internalization status when treated with 30 µg Fe/mL. The quantification by ICP-MS evaluate the stem cells homing by identifying 3.95 ± 0.37 µg Fe/mL in the control and 6.61 ± 0.84 µg Fe/mL in the bone marrow of transplanted animals. In addition, 2.14 ± 0.66 mg Fe/g in the spleen of the control group and 2.17 ± 0.59 mg Fe/g in the spleen of the experimental group was also measured. Moreover, the bioluminescence imaging provided the follow up on the hematopoietic stem cells behavior by monitoring their distribution by the bioluminescence signal. Lastly, the blood count enabled the monitoring of animal hematopoietic reconstitution and ensured the transplantation effectiveness.

Nucleated red blood cell: a feasible quality parameter of cord blood units.

INTRODUCTION: Umbilical cord blood is an alternative source of hematopoietic progenitor cells for bone marrow transplantation; however, it is associated with a higher graft failure rate. The presence of a high rate of nucleated red blood cells (NRBCs) seems to be related to a greater capacity for engraftment, although is also associated with fetal distress conditions. We analyzed the correlation of the NRBC with quality parameters and its association with the utilization score of a cord blood unit. STUDY DESIGN AND METHOD: Data of 3346 units collected in a public cord blood bank from May 2010 to December 2017 were analyzed, retrospectively, to identify factors associated with an increased number of nucleated red blood cells and its correlation with the engraftment capacity measured through total nucleated cells (TNCs) and CD34 positive cells. We also evaluated the utilization score of these units and identified an NRBC cutoff associated with a higher score. RESULTS: The median volume collected was 104 mL (42-255), the pre-processing TNC count was 144.77 × 107 (95.46-477.18), the post-processing TNC count was 119.44 × 107 (42.7-477.18), the CD34 count was 4.67 × 106 (0.31-48.01), the NRBC count was 5 (0-202) and the utilization score was 0.0228 (0.00143-0.9740). The NRBC showed a correlation with the collected volume, TNC and CD34 positive cells and a higher utilization score and the receiver operating characteristic (ROC) curve analysis identified the five NRBC/100 leukocytes cutoff that correlates better with the probability of use. No association with pathological conditions and the NRBC rate was observed. CONCLUSIONS: The NRBC is a feasible parameter for the screening of the cord blood unit (CBU) and the minimum cutoff of five NRBC/100 leukocytes can be a strategy in conjunction with the TNC to identify better units for cord blood bank sustainability.

How mesenchymal stem cell cotransplantation with hematopoietic stem cells can improve engraftment in animal models.

BACKGROUND: Bone marrow transplantation (BMT) can be applied to both hematopoietic and nonhematopoietic diseases; nonetheless, it still comes with a number of challenges and limitations that contribute to treatment failure. Bearing this in mind, a possible way to increase the success rate of BMT would be cotransplantation of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) to improve the bone marrow niche and secrete molecules that enhance the hematopoietic engraftment. AIM: To analyze HSC and MSC characteristics and their interactions through cotransplantation in murine models. METHODS: We searched for original articles indexed in PubMed and Scopus during the last decade that used HSC and MSC cotransplantation and in vivo BMT in animal models while evaluating cell engraftment. We excluded in vitro studies or studies that involved graft versus host disease or other hematological diseases and publications in languages other than English. In PubMed, we initially identified 555 articles and after selection, only 12 were chosen. In Scopus, 2010 were identified, and six were left after the screening and eligibility process. RESULTS: Of the 2565 articles found in the databases, only 18 original studies met the eligibility criteria. HSC distribution by source showed similar ratios, with human umbilical cord blood or animal bone marrow being administered mainly with a dose of 1 × 107 cells by intravenous or intrabone routes. However, MSCs had a high prevalence of human donors with a variety of sources (umbilical cord blood, bone marrow, tonsil, adipose tissue or fetal lung), using a lower dose, mainly 106 cells and ranging 104 to 1.5 × 107 cells, utilizing the same routes. MSCs were characterized prior to administration in almost every experiment. The recipient used was mostly immunodeficient mice submitted to low-dose irradiation or chemotherapy. The main technique of engraftment for HSC and MSC cotransplantation evaluation was chimerism, followed by hematopoietic reconstitution and survival analysis. Besides the engraftment, homing and cellularity were also evaluated in some studies. CONCLUSION: The preclinical findings validate the potential of MSCs to enable HSC engraftment in vivo in both xenogeneic and allogeneic hematopoietic cell transplantation animal models, in the absence of toxicity.

Improved engraftment and therapeutic efficacy by human genome-edited hematopoietic stem cells with Busulfan-based myeloablation.

Autologous hematopoietic stem cell transplantation using genome-edited cells can become a definitive therapy for hematological and non-hematological disorders with neurological involvement. Proof-of-concept studies using human genome-edited hematopoietic stem cells have been hindered by the low efficiency of engraftment of the edited cells in the bone marrow and their modest efficacy in the CNS. To address these challenges, we tested a myeloablative conditioning regimen based on Busulfan in an immunocompromised model of mucopolysaccharidosis type 1. Compared with sub-lethal irradiation, Busulfan conditioning enhanced the engraftment of edited CD34+ cells in the bone marrow, as well the long-term homing and survival of bone-marrow-derived cells in viscera, and in the CNS, resulting in higher transgene expression and biochemical correction in these organs. Edited cell selection using a clinically compatible marker resulted in a population with low engraftment potential. We conclude that conditioning can impact the engraftment of edited hematopoietic stem cells. Furthermore, Busulfan-conditioned recipients have a higher expression of therapeutic proteins in target organs, particularly in the CNS, constituting a better conditioning approach for non-hematological diseases with neurological involvement.

Hepatitis B virus among hematopoietic stem cell transplant recipients: Antiviral impact in seroconversion, engraftment, and mortality in a Latin American center.

BACKGROUND: Hepatitis B virus (HBV) infection is a worldwide concern with a broad distribution. In immunosuppressed populations, such as solid organ and hematopoietic stem cell transplant (HSCT) recipients, it can reactivate leading to acute hepatic failure. Different risk factors are known for higher rates of reactivation, and entecavir, tenofovir, and lamivudine are often used for prophylaxis and treatment. However, data regarding the impact of antiviral drugs in neutrophil and platelet engraftment are still unknown and concern the management of viral hepatitis post-HSCT. METHODS: We performed a single-center, retrospective, observational study reviewing medical records of patients referred for hematopoietic stem cell transplant from 2010 to 2017, which were also HBV infected, aiming to describe outcomes related to antiviral treatment and also the impact on platelet and neutrophil recovery after transplant. A secondary goal consisted of analyzing the impact of HBV infection in early and late mortality post-HSCT. The study included patients with positive blood bank screening for hepatitis B infection (HBsAg, Anti-HBc or HBV-NAT), confirmed later on by a laboratory routine serology. RESULTS: A total of 1132 hematopoietic stem cell recipients were assessed between 2010 and 2017. Eighty-six patients were confirmed to have HBV infection, of which six were HBsAg-positive, 20 were isolated anti-HBc-positive, and 60 had resolved infection (anti-HBc-positive and anti-HBs-positive). With regard to prophylaxis, 19 patients underwent HSCT on HBV antiviral therapy or prophylaxis: two were HBeAg-positive, three were HBeAg-negative and HBV-DNA was only detectable in three of them. Moreover, one patient had an occult HBV infection. Regarding therapy, 9 patients were on entecavir, 6 patients on lamivudine, two on tenofovir, and two of them on a combination of tenofovir + lamivudine due to HIV co-infection. Reverse seroconversion was not identified in any patients receiving antiviral therapy or prophylaxis, but it was detected in one patient with occult hepatitis B and another with resolved infection. No severe side effects led to therapy discontinuation in the treated group, which also did not have any significant delay in neutrophil or platelet engraftment when compared to patients without antiviral therapy. In addition, the only factors associated with increased mortality were transplant onset after 50 years, allogeneic transplant and myeloablative conditioning regimens. Interestingly, the presence of HBsAg or detectable HBV-DNA was not related to worse outcomes, neither the use of rituximab. In multivariate analysis, the use of antiviral therapy, the occurrence of graft-versus-host disease or CMV reactivation also was not linked to increased mortality. CONCLUSIONS: To sum up, HBV serology, ALT, and HBV-DNA monitoring are essential to detect hepatic flares earlier, even in populations with chronic inactive hepatitis, due to the possibility of later seroconversion. HBV infection was not related to increased 2-year mortality post-transplant. Antiviral prophylaxis did not cause any important clinical or laboratory side effects that could demand discontinuation, and its use was not associated with later neutrophil and platelet engraftments.

Engraftment of Human Stem Cell-Derived Otic Progenitors in the Damaged Cochlea.

Most cases of sensorineural deafness are caused by degeneration of hair cells. Although stem/progenitor cell therapy is becoming a promising treatment strategy in a variety of organ systems, cell engraftment in the adult mammalian cochlea has not yet been demonstrated. In this study, we generated human otic progenitor cells (hOPCs) from induced pluripotent stem cells (iPSCs) in vitro and identified these cells by the expression of known otic markers. We showed successful cell transplantation of iPSC-derived-hOPCs in an in vivo adult guinea pig model of ototoxicity. The delivered hOPCs migrated throughout the cochlea, engrafted in non-sensory regions, and survived up to 4 weeks post-transplantation. Some of the engrafted hOPCs responded to environmental cues within the cochlear sensory epithelium and displayed molecular features of early sensory differentiation. We confirmed these results with hair cell progenitors derived from Atoh1-GFP mice as donor cells. These mouse otic progenitors transplanted using the same in vivo delivery system migrated into damaged cochlear sensory epithelium and adopted a partial sensory cell fate. This is the first report of the survival and differentiation of hOPCs in ototoxic-injured mature cochlear epithelium, and it should stimulate further research into cell-based therapies for treatment of deafness.

PAI-1 inhibition by simvastatin as a positive adjuvant in cell therapy.

Cell therapy (CT) can be briefly described as the use of cells or cell components in the treatment of diseases. One of the main challenges in establishing new cell types for therapy is the low survival rates of homing cells. Glycoprotein plasminogen activator inhibitor 1 (PAI-1) is a key regulator of the plasminogen activation system, and also an essential mediator of mesenchymal stem cell (MSC) post-transplant survival rate in the target tissue. It was previously observed that the survival of cells infused into the transplanted tissue increase in the presence of PAI-1 neutralizing antibodies. Simvastatin acts at several levels in the protein cascade regulating PAI-1 levels. Thus, simvastatin-induced reduction of PAI-1 levels has a therapeutic potential by modulating the main processes involved in the creation of an inhospitable environment during the process of injury (fibrosis and cell migration). In this way, simvastatin modulates process such as migration, that plays a key role in homing and engraftment of cells after cell therapy. Due to this modulatory effect, research groups proposed the use of simvastatin as an adjuvant in different cell therapy approaches. These observations allow the proposition of the potential use of simvastatin, and possibly other statins, as an adjuvant in cell therapy, due to a mechanism of action that acts in the tissue microenvironment, promoting a better efficiency of the homing and, as a consequence, an enhancement of the paracrine effects of the stem cells in the process of tissue regeneration.

Effects of omega-3 on matrix metalloproteinase-9, myoblast transplantation and satellite cell activation in dystrophin-deficient muscle fibers.

In Duchenne muscular dystrophy (DMD), lack of dystrophin leads to progressive muscle degeneration, with DMD patients suffering from cardiorespiratory failure. Cell therapy is an alternative to life-long corticoid therapy. Satellite cells, the stem cells of skeletal muscles, do not completely compensate for the muscle damage in dystrophic muscles. Elevated levels of proinflammatory and profibrotic factors, such as metalloproteinase 9 (MMP-9), impair muscle regeneration, leading to extensive fibrosis and poor results with myoblast transplantation therapies. Omega-3 is an anti-inflammatory drug that protects against muscle degeneration in the mdx mouse model of DMD. In the present study, we test our hypothesis that omega-3 affects MMP-9 and thereby benefits muscle regeneration and myoblast transplantation in the mdx mouse. We observe that omega-3 reduces MMP-9 gene expression and improves myoblast engraftment, satellite cell activation, and muscle regeneration by mechanisms involving, at least in part, the regulation of macrophages, as shown here with the fluorescence-activated cell sorting technique. The present study demonstrates the benefits of omega-3 on satellite cell survival and muscle regeneration, further supporting its use in clinical trials and cell therapies in DMD.

Two complementary strategies to improve cell engraftment in mesenchymal stem cell-based therapy: Increasing transplanted cell resistance and increasing tissue receptivity.

Over the past 2 decades, therapies based on mesenchymal stem cells (MSC) have been tested to treat several types of diseases in clinical studies, due to their potential for tissue repair and regeneration. Currently, MSC-based therapy is considered a biologically safe procedure, with the therapeutic results being very promising. However, the benefits of these therapies are not stable in the long term, and the final outcomes manifest with high inter-patient variability. The major cause of these therapeutic limitations results from the poor engraftment of the transplanted cells. Researchers have developed separate strategies to improve MSC engraftment. One strategy aims at increasing the survival of the transplanted MSCs in the recipient tissue, rendering them more resistant to the hostile microenvironment (cell-preconditioning). Another strategy aims at making the damaged tissue more receptive to the transplanted cells, favoring their interactions (tissue-preconditioning). In this review, we summarize several approaches using these strategies, providing an integral and updated view of the recent developments in MSC-based therapies. In addition, we propose that the combined use of these different conditioning strategies could accelerate the process to translate experimental evidences from pre-clinic studies to the daily clinical practice.

Specific factors influence the success of autologous and allogeneic hematopoietic stem cell transplantation.

Successful hematopoietic stem cell transplantation (HSCT), both autologous and allogeneic, requires a rapid and durable engraftment, with neutrophil (>500/microL) and platelet (>20,000/microL) reconstitution. Factors influencing engraftment after autologous or allogeneic HSCT were investigated in 65 patients: 25 autologous peripheral stem cell transplantation (PBSCT) and 40 allogeneic bone marrow transplantation (BMT) patients. The major factor affecting engraftment was the graft source for HSCT. Neutrophil and platelet recovery were more rapid in autologous PBSCT than in allogeneic BMT [neutrophil occurring in median on day 10.00 (09.00/11.00) and 19.00 (16.00/23.00) and platelet on day 11.00 (10.00/13.00) and 21.00 (18.00/25.00), respectively; p < 0.0001]. The type of disease also affected engraftment, where multiple myeloma (MM) and lymphoma showed faster engraftment when compared with leukemia, syndrome myelodysplastic (SMD) and aplastic anemia (AA) and MM presented the best overall survival (OS) in a period of 12 months. Other factors included the drug used in the conditioning regimen (CR), where CBV, melphalan (M-200) and FluCy showed faster engraftment and M-200 presented the best OS, in a period of 12 months and age, where 50-59 years demonstrated faster engraftment. Sex did not influence neutrophil and platelet recovery.