Cell reprogramming shapes the mitochondrial DNA landscape

Individual induced pluripotent stem cells (iPSCs) show considerable phenotypic heterogeneity, but the reasons for this are not fully understood. Comprehensively analysing the mitochondrial genome (mtDNA) in 146 iPSC and fibroblast lines from 151 donors, we show that most age-related fibroblast mtDNA mutations are lost during reprogramming. However, iPSC-specific mutations are seen in 76.6% (108/141) of iPSC lines at a mutation rate of 8.62 × 10−5/base pair. The mutations observed in iPSC lines affect a higher proportion of mtDNA molecules, favouring non-synonymous protein-coding and tRNA variants, including known disease-causing mutations. Analysing 11,538 single cells shows stable heteroplasmy in sub-clones derived from the original donor during differentiation, with mtDNA variants influencing the expression of key genes involved in mitochondrial metabolism and epidermal cell differentiation. Thus, the dynamic mtDNA landscape contributes to the heterogeneity of human iPSCs and should be considered when using reprogrammed cells experimentally or as a therapy.

Noninvasive Assessment of Exosome Pharmacokinetics In Vivo: A Review

Exosomes, intraluminal vesicles that contain informative DNA, RNA, proteins, and lipid membranes derived from the original donor cells, have recently been introduced to therapy and diagnosis. With their emergence as an alternative to cell therapy and having undergone clinical trials, proper analytical standards for evaluating their pharmacokinetics must now be established. Molecular imaging techniques such as fluorescence imaging, magnetic resonance imaging, and positron emission tomography (PET) are helpful to visualizing the absorption, distribution, metabolism, and excretion of exosomes. After exosomes labelled with a fluorescer or radioisotope are administered in vivo, they are differentially distributed according to the characteristics of each tissue or lesion, and real-time biodistribution of exosomes can be noninvasively monitored. Quantitative analysis of exosome concentration in biological fluid or tissue samples is also needed for the clinical application and industrialization of exosomes. In this review, we will discuss recent pharmacokinetic applications to exosomes, including labelling methods for in vivo imaging and analytical methods for quantifying exosomes, which will be helpful for evaluating pharmacokinetics of exosomes and improving exosome development and therapy.

Will you give my kidney back? Organ restitution in living-related kidney transplantation: ethical analyses

In this article, we perform a thought experiment about living donor kidney transplantation. If a living kidney donor becomes in need of renal replacement treatment due to dysfunction of the remaining kidney after donation, can the donor ask the recipient to give back the kidney that had been donated? We call this problem organ restitution and discussed it from the ethical viewpoint. Living organ transplantation is a kind of ‘designated donation’ and subsequently has a contract-like character. First, assuming a case in which original donor (A) wishes the return of the organ which had been transplanted into B, and the original recipient (B) agrees, organ restitution will be permissible based on contract-like agreement. However, careful and detailed consideration is necessary to determine whether this leaves no room to question the authenticity of B’s consent. Second, if B offers to give back the organ to A, then B’s act is a supererogatory act, and is praiseworthy and meritorious. Such an offer is a matter of virtue, not obligation. Third, if A wishes B to return the organ, but B does not wish/allow this to happen, it is likely difficult to justify returning the organ to A by violating B’s right to bodily integrity. But B’s refusal to return the donated organ cannot be deemed praiseworthy, because B forgets the great kindness once received from A. Rather than calling this an obligation, we encourage B to consider such virtuous conduct.

T-Cell Depleted (TCD) Hematopoietic Stem Cell (HSC) Boosts From the Original Donor without Conditioning for Poor Graft Function (PGF) Following Allogeneic Hematopoietic Stem Cell Transplants (HSCT)

Abstract 1942 BACKGROUND: Poor graft function without immune rejection, defined as persistent cytopenias with a hypocellular marrow and full donor chimerism, is a life-threatening complication after allogeneic HSCT. Treatment options include supportive therapy with transfusions and growth factors, vs administration of additional HSCs from the same donor without any conditioning (stem cell boost). The use of unmanipulated boosts increases the risk of development or worsening of GvHD by virtue of infusing mature T cells. The aim of this retrospective study was to evaluate the efficacy and safety of TCD boosts in patients with PGF. PATIENT AND METHODS: Between January 1992 and December 2007, 35 patients from a single center with PGF following either an unmanipulated (10) or a TCD (25) allogeneic HCST received a TCD HSC boost collected from the original donor. T cells were removed ex-vivo from marrow grafts with soybean lectin agglutinin and sheep red blood cells (sRBC) rosetting and from peripheral blood stem cell grafts by positive selection with a CD34 antibody (Isolex) followed by sRBC rosetting. HSC donors were matched related (21), mismatched related (5) and unrelated (9). Indication for first transplant included: aplastic anemia (2), non-Hodgkin's lymphoma (5) and myeloid malignancies (28). The preparative regimen was myeloablative for all recipients of unmanipulated grafts and for 22 of 25 recipients of TCD grafts. With the exception of one patient, the cell dose as measured by total nucleated cell dose/kg or CD34 cells/kg was adequate. Following the initial transplant, all patients had partial or complete recovery of blood counts and full donor chimerism documented by karyotype, FISH analysis or DNA polymorphism. The median time from first transplant to the diagnosis of PGF was 4.5 months (0.72-90.7). Etiologies of PGF included: viral infection and anti-virals (10), bacterial sepsis (3), Mycobacterial infection (4), low cell dose (1), GvHD 12, and idiopathic (5). None of the patients had evidence of relapse or progression of their underlying disease. All patients received a TCD boost from the original donor; with 21 patients receiving bone marrow and 14 receiving PB HSC. RESULTS: Seven patients died before day 21 post boost and were not evaluable for blood count improvement. Improvement (PLT>50,000/μL, ANC>500/μL) occurred in 20 of the 28 evaluable patients (71.4 %) at a median time of 3.2 months; 17 of them (60.7%) had a more substantial improvement (PLT ≥100,000/μL and ANC31000/μL) at a median of 8.81 months. TCD boost infusions were well tolerated with no significant adverse events. Also, no new onset GvHD occurred after TCD boosts; although two patients with preexisting GvHD flared. The median survival for all patients following TCD boosts was 21.47 months (range: 1.84–208.45). The 2-year and 5-year survivals were 48.6% and 37.1% respectively. The 2-year survival for patients who had improvement of their counts was 90% and for those who remained pancytopenic despite the boost was 18%. The 2-year survival according to etiology was 52.9% for the infection group, 50% for the idiopathic and low cell dose, and 33.3% for the GvHD group. Patients were also analyzed according to their medical condition prior to receiving the boost and were separated into four groups based on organ function (serum creatinine ≥ 2; total bilirubin ≥ 2; need for mechanical ventilation) and infection status. Group A: outpatient with no infection and no organ dysfunction (9); group B: hospitalized but afebrile with no infection or organ dysfunction (7); group C: febrile or documented infection but preserved organ function (8); group D: organ dysfunction with or without a concurrent infection (11). The 2-year survival for each group was 55.6%, 85.7%, 50%, and 9%, respectively. Patients with organ dysfunction with or without concurrent infection had the lowest survival. Causes of death included: GvHD (10), infection (5), relapse (4), organ failure (3), and poor graft function (1). CONCLUSION: Treatment of PGF with a TCD HSC boost from the original donor is safe and effective with minimal risk of GvHD. Medical status at the time of the boost infusion had a significant impact on outcome. A TCD boost should be considered early in the course of PGF as once complications of persistently low blood counts occur the potential for benefit sharply declines. Disclosures: Small: Pfizer, Inc: Equity Ownership, family member employed by Pfizer, Inc. Perales:Pfizer, Inc: Equity Ownership.