A novel autologous stem cell procedure for the treatment of aplastic anaemia using reprogrammed mature adult cells: a pilot study.

BACKGROUND & OBJECTIVES: Aplastic anaemia is a life threatening rare bone marrow failure disorder. The underlying haematopoietic cellular deficit leads to haemorrhage, infection and severe anaemia. The treatment of choice for this haematological condition is allogeneic bone marrow transplantation from fully matched HLA sibling. Though this procedure is curative in the majority of young patients with aplastic anaemia, extending this benefit to older patients or those lacking a family donor remains a major challenge. Herein, the safety and efficacy of infusing autologous retrodifferentiated haematopoietic stem cells (RHSC) into four patients with aplastic anaemia without the use of any pre- or post-conditioning regimen including immunosuppression is described. METHODS: Un-mobilized, mononuclear cells were harvested from four patients with acquired aplastic anaemia by aphaeresis. Mononuclear cells of patients were cultured with purified monoclonal antibody against the monomorphic regions of the beta chain of MHC class II antigens (Clone CR3/43) for 3 h, to obtain autologous RHSC. Autologous RHSC were washed and infused into the four patients without the use of any pre- or post-conditioning regimen. Thereafter, the efficacy (engraftment) of autologous RHSC was assessed in these patients. RESULTS: Following single infusion of the autologous RHSC, two of the four patients with aplastic anaemia become transfusion independent for more than seven years. Karyotyping and G-banding analysis prior and post-procedure in all patients remained the same. INTERPRETATION & CONCLUSIONS: The findings of this pilot study demonstrated the functional utility of reprogrammed fully differentiated adult cells into pluripotent stem cells with extensive repopulation potentials in a human setting and without any pre- or post-conditioning regimen, including immunosuppression. This autologous approach of stem cell creation may broaden the curative potentials of stem cell therapy to a wider population of patients with aplastic anaemia, including many patients suffering from other haematological and non-haematological disorders.

Infusion of autologous retrodifferentiated stem cells into patients with beta-thalassemia.

Beta-thalassemia is a genetic, red blood cell disorder affecting the beta-globin chain of the adult hemoglobin gene. This results in excess accumulation of unpaired alpha-chain gene products leading to reduced red blood cell life span and the development of severe anemia. Current treatment of this disease involves regular blood transfusion and adjunct chelation therapy to lower blood transfusion-induced iron overload. Fetal hemoglobin switching agents have been proposed to treat genetic blood disorders, such as sickle cell anemia and beta-thalassemia, in an effort to compensate for the dysfunctional form of the beta-globin chain in adult hemoglobin. The rationale behind this approach is to pair the excess normal alpha-globin chain with the alternative fetal gamma-chain to promote red blood cell survival and ameliorate the anemia. Reprogramming of differentiation in intact, mature, adult white blood cells in response to inclusion of monoclonal antibody CR3/43 has been described. This form of retrograde development has been termed "retrodifferentiation", with the ability to re-express a variety of stem cell markers in a heterogeneous population of white blood cells. This form of reprogramming, or reontogeny, to a more pluripotent stem cell state ought to recapitulate early hematopoiesis and facilitate expression of a fetal and/or adult program of hemoglobin synthesis or regeneration on infusion and subsequent redifferentiation. Herein, the outcome of infusion of autologous retrodifferentiated stem cells (RSC) into 21 patients with beta-thalassemia is described. Over 6 months, Infusion of 3-h autologous RSC subjected to hematopoietic-conducive conditions into patients with beta-thalassemia reduced mean blood transfusion requirement, increased mean fetal hemoglobin synthesis, and significantly lowered mean serum ferritin. This was always accompanied by an increase in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) in such patients. No adverse side effects in response to the infusion of autologous RSC were noted. This novel clinical procedure may profoundly modify the devastating course of many genetic disorders in an autologous setting, thus paving the way to harnessing pluripotency from differentiated cells to regenerate transiently an otherwise genetically degenerate tissue such as thalassemic blood.

Harnessing pluripotency from differentiated cells: a regenerative source for tissue-specific stem cell therapies.

Processes involving conversion of mature adult cells into undifferentiated cells have tremendous therapeutic potential in treating a variety of malignant and non-malignant disorders, including degenerative diseases. This can be achieved in autologous or allogeneic settings, by replacing either defective cells or regenerating those that are in deficit through reprogramming more committed cells into stem cells. The concept behind reprogramming differentiated cells to a stem cell state is to enable the switching of development towards the required cell lineage that is capable of correcting the underlying cellular dysfunction. The techniques by which differentiated cells can reverse their development, become pluripotent stem cells and transdifferentiate to give rise to new tissue or an entire organism are currently under intense investigation. Examples of reprogramming differentiation in mature adult cells include nuclear reprogramming of more committed cells using the cytoplasm of empty oocytes obtained from a variety of animal species, or cell surface contact of differentiated cells through receptor ligand interaction. Such ligands include monoclonal antibodies, cytokines or synthetic chemical compounds. Despite controversies surrounding such techniques, the concept behind identification and design/screening of biological or pharmacological compounds to enable re-switching of cell fate in-vivo or ex-vivo is paramount for current drug therapies to be able to target more specifically cellular dysfunction at the tissue/organ level. Herein, this review discusses current research in cellular reprogramming and its potential application in regenerative medicine.

Induction of stem cell-like plasticity in mononuclear cells derived from unmobilised adult human peripheral blood.

Undifferentiated pluripotent stem cells with flexible developmental potentials are not normally found in peripheral blood. However, such cells have recently been reported to reside in the bone marrow. Herein are reported methods of inducing pluripotency in cells derived from unmobilised adult human peripheral blood. In response to the inclusion of purified CR3/43 monoclonal antibody (mAb) to well-established culture conditions, mononuclear cells (MNC) obtained from a single blood donor are converted into pluripotent haematopoietic, neuronal and cardiomyogenic progenitor stem cells or undifferentiated stem cells. The haematopoietic stem cells are CD34+, clonogenic and have been shown to repopulate non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The neuronal precursors transcribe the primitive stem cell markers OCT-4 and nestin, and on maturation, differentially stain positive for neuronal, glial or oligodendrocyte-specific antigens. The cardiomyogenic progenitor stem cells form large bodies of asynchronously beating cells and differentiate into mature cardiomyocytes which transcribe GATA-4. The undifferentiated stem cells do not express haematopoietic-associated markers, are negative for major histocompatibility complex (MHC) class I and II antigens, transcribe high levels of OCT-4 and form embryoid body (EB)-like structures. This induction of stem cell-like plasticity in MNC may have proceeded by a process of retrodifferentiation but, in any case, could have profound clinical and pharmacological implications. Finally, the flexibility and the speed by which a variety of stem cell classes can be generated ex vivo from donor blood could potentially transfer this novel process into a less invasive automated clinical procedure.