Colin P. McGuckin

Production of stem cells with embryonic characteristics from human umbilical cord blood

Abstract.  When will embryonic stem cells reach the clinic? The answer is simple – not soon! To produce large quantities of homogeneous tissue for transplantation, without feeder layers, and with the appropriate recipients immunological phenotype, is a significant scientific hindrance, although adult stem (ADS) cells provide an alternative, more ethically acceptable, source. The annual global 100 million human birth rate proposes umbilical cord blood (UCB) as the largest untouched stem cell source, with advantages of naive immune status and relatively unshortened telomere length. Here, we report the worlds first reproducible production of cells expressing embryonic stem cell markers, – cord‐blood‐derived embryonic‐like stem cells (CBEs). UCB, after elective birth by Caesarean section, has been separated by sequential immunomagnetic removal of nucleate granulocytes, erythrocytes and haemopoietic myeloid/lymphoid progenitors. After 7 days of high density culture in microflasks, (105 cells/ml, IMDM, FCS 10%, thrombopoietin 10 ng/ml, flt3‐ligand 50 ng/ml, c‐kit ligand 20 ng/ml). CBE colonies formed adherent to the substrata; these were maintained for 6 weeks, then were subcultured and continued for a minimum 13 weeks. CBEs were positive for TRA‐1‐60, TRA‐1‐81, SSEA‐4, SSEA‐3 and Oct‐4, but not SSEA‐1, indicative of restriction in the human stem cell compartment. The CBEs were also microgravity–bioreactor cultured with hepatocyte growth medium (IMDM, FCS 10%, HGF 20 ng/ml, bFGF 10 ng/ml, EGF 10 ng/ml, c‐kit ligand 10 ng/ml). After 4 weeks the cells were found to express characteristic hepatic markers, cytokeratin‐18, α‐foetoprotein and albumin. Thus, such CBEs are a viable human alternative from embryonic stem cells for stem cell research, without ethical constraint and with potential for clinical applications.

Diamond‐Blackfan anaemia in the U.K.: analysis of 80 cases from a 20‐year birth cohort

The U.K. Diamond‐Blackfan Anaemia (DBA) Registry was established with the aim of providing a representative database for studies on the aetiology, pathophysiology and treatment of DBA. We have analysed retrospective data from 80 cases (33 male, 47 female) born in the U.K. in a 20‐year period (1975–94), representing an annual incidence of 5 per million live births. Ten children from seven families had an apparently familial disorder. 13% were anaemic at birth, and 72.5% had presented by the age of 3 months. 67% had macrocytosis at presentation. 72% responded initially to steroids, and at the time of study 61% were transfusion‐independent (45% steroid‐dependent) and 39% required regular transfusions. Unequivocal physical anomalies, predominantly craniofacial, were present in 37%, and were more likely in boys (52%) than girls (25%). 18% had thumb abnormalities. Height was below the third centile for age in 28%, and 31% had neither short stature nor physical anomalies. Four children without physical abnormalities had normal red cell indices, and achieved steroid‐independent remission, suggesting transient erythroblastopenia of childhood rather than DBA. The birth month distribution of children with sporadic DBA and craniofacial dysmorphism showed a possible seasonality, consistent with a viral aetiology.

Characterization of a Lineage-Negative Stem-Progenitor Cell Population Optimized for Ex Vivo Expansion and Enriched for LTC-IC

Current hematopoietic stem cell transplantation protocols rely heavily upon CD34+ cells to estimate hematopoietic stem and progenitor cell (HSPC) yield. We and others previously reported CD133+ cells to represent a more primitive cell population than their CD34+ counterparts. However, both CD34+ and CD133+ cells still encompass cells at various stages of maturation, possibly impairing long‐term marrow engraftment. Recent studies demonstrated that cells lacking CD34 and hematopoietic lineage markers have the potential of reconstituting long‐term in vivo hematopoiesis. We report here an optimized, rapid negative‐isolation method that depletes umbilical cord blood (UCB) mononucleated cells (MNC) from cells expressing hematopoietic markers (CD45, glycophorin‐A, CD38, CD7, CD33, CD56, CD16, CD3, and CD2) and isolates a discrete lineage‐negative (Lin−) cell population (0.10% ± 0.02% MNC, n = 12). This primitive Lin− cell population encompassed CD34+/− and CD133+/− HSPC and was also enriched for surface markers involved in HSPC migration, adhesion, and homing to the bone marrow (CD164, CD162, and CXCR4). Moreover, our depletion method resulted in Lin− cells being highly enriched for long‐term culture‐initiating cells when compared with both CD133+ cells and MNC. Furthermore, over 8 weeks in liquid culture stimulated by a cytokine cocktail optimized for HSPC expansion, TPOFLK (thrombopoietin 10 ng/ml, Flt3 ligand 50 ng/ml, c‐Kit ligand 20 ng/ml) Lin− cells underwent slow proliferation but maintained/expanded more primitive HSPC than CD133+ cells. Therefore, our Lin− stem cell offers a promising alternative to current HSPC selection methods. Additionally, this work provides an optimized and well‐characterized cell population for expansion of UCB for a wider therapeutic potential, including adult stem cell transplantation.

Multiparametric analysis of immature cell populations in umbilical cord blood and bone marrow.

Abstract: Adult stem cells are finding increased therapeutic potential not least in tissue regeneration protocols. The cell sources being proposed for such protocols include embryonic, umbilical cord blood (CB) and adult bone marrow (BM). Although embryonic sources are controversial, CB and marrow are available immediately. The appropriate cells of use in these sources are considered to be extremely rare and a characterisation of the starting cell source is important for the development of adult stem cell protocols and ex vivo expansion. Umbilical CB and BM mononuclear cells were labelled for the antigens CD34, CD133, CD117, CD164, Thy‐1 or CD38, and additional intracellular CD34 antigen. Three dimensional flow‐cytometric analyses were carried out together with dual laser confocal microscope analysis for antigen profile expression. Variable levels of immaturity were detected on CB and BM populations using internal and external CD34 antigen. For CB and BM cells, internal CD34 (intCD34+) could be detected on co‐expressing CD133+ cells before expression of external CD34 antigen (extCD34+). CD38 co‐expression analysis also showed that a small but distinct group of cells expressing low CD38 and no external CD34 antigen could be detected. Additional phenotyping of these cells using CD117, Thy‐1, CD164 and CD133 demonstrated variable primitive status detectable within the external CD34− population. Newly harvested primary CB and BM populations were shown to contain not only cellular populations of known standard sequential maturity but also populations of more extreme rarity. The presence of cells which lacked extracellular CD34 antigen, in both CB and BM, but which possessed CD133, has important implications for purification of human stem cells in clinical applications.

AC133+ umbilical cord blood progenitors demonstrate rapid self-renewal and low apoptosis.

Summary. Umbilical cord blood (UCB) provides immediate access to haemopoietic stem/progenitor cells (HSPC) but low cell number restricts use in full adult bone marrow reconstitution. This study investigated early ex vivo expansion kinetics of UCB AC133+ cells (2–4 × 104/ml), mononuclear cells (MNC, 1–2 × 106/ml) and AC133negative cells (AC133neg, 2–4 × 104/ml) in stroma‐free 8 d liquid culture (fetal bovine serum‐supplemented Iscoves‐modified Dulbeccos medium (IMDM) with either ‘K36EG’[c‐Kit ligand, interleukin 3 (IL‐3), IL‐6, erythropoietin, granulocyte colony‐stimulating factor] or ‘TPOFL’ (thrombopoietin, Flt‐3 ligand). Cell enumeration, apoptosis assay and AC133/CD34/CD38 antigen immunophenotyping were performed at d 0, 3, 5 and 8. All three cell populations went through a proliferation lag phase between d 3 and d 5. AC133+ cells recovered better from lag phase with significantly higher fold increase (FI) when compared with MNC and AC133neg populations (K36EG FI: 15·04 ± 5·46; TPOFL FI: 8·59 ± 2·92, P < 0·05). After 8 d, populations lacking AC133+ cells were significantly more inclined to undergo apoptosis under proliferative conditions (P < 0·01). Also, when compared with K36EG, 8 d TPOFL‐expanded AC133+ cells encompassed a significantly higher percentage of AC133+ and CD34+ early HSPC (K36EG: 20·50 ± 2·36; TPOFL: 47·00 ± 7·69; P < 0·05). In conclusion, TPOFL synergism demonstrated the potential for AC133+ HSPC ex vivo expansion inducing self‐renewal, early HSPC maintenance and promoting cell survival status.

Robust and automated unimodal histogram thresholding and potential applications

In this paper, three new histogram-based algorithms are presented to segment images expressing unimodal intensity histograms. These algorithms are applied to laser scanning confocal microscope images (known to often exhibit unimodal histograms) to identify fluorescent signals, and other applications are also shown. The first algorithm facilitates linear diffusion to investigate dynamic histogram features in scale-space. The second algorithm is based on a histogram comparison between a reference area and the whole image at reduced scale. The third algorithm uses the maximisation of a between-class variance criterion applied to image histograms. Results obtained from automatic thresholding of confocal microscopy images show good agreement between the algorithms. Further applications to segment other images are also shown.

New perspectives in stem cell research: beyond embryonic stem cells

Although stem cell research is a rather new field in modern medicine, media soon popularized it. The reason for this hype lies in the potential of stem cells to drastically increase quality of life through repairing aging and diseased organs. Nevertheless, the essence of stem cell research is to understand how tissues are maintained during adult life. In this article, we summarize the various types of stem cells and their differentiation potential in vivo and in vitro. We review current clinical applications of stem cells and highlight problems encountered when going from animal studies to clinical practice. Furthermore, we describe the current state of induced pluripotent stem cell technology and applications for disease modelling and cell replacement therapy.

Ischemic brain injury: a consortium analysis of key factors involved in mesenchymal stem cell-mediated inflammatory reduction.

Increasing global birth rate, coupled with the aging population surviving into their eighth decade has lead to increased incidence diseases, hitherto designated as rare. Brain related ischemia, at birth, or later in life, during, for example stroke, is increasing in global prevalence. Reactive microglia can contribute to neuronal damage as well as compromising transplantion. One potential treatment strategy is cellular therapy, using mesenchymal stem cells (hMSCs), which possess immunomodulatory and cell repair properties. For effective clinical therapy, mechanisms of action must be understood better. Here multicentre international laboratories assessed this question together investigating application of hMSCs neural involvement, with interest in the role of reactive microglia. Modulation by hMSCs in our in vivo and in vitro study shows they decrease markers of microglial activation (lower ED1 and Iba) and astrogliosis (lower GFAP) following transplantation in an ouabain-induced brain ischemia rat model and in organotypic hippocampal cultures. The anti-inflammatory effect in vitro was demonstrated to be CD200 ligand dependent with ligand expression shown to be increased by IL-4 stimulation. hMSC transplant reduced rat microglial STAT3 gene expression and reduced activation of Y705 phosphorylated STAT3, but STAT3 in the hMSCs themselves was elevated upon grafting. Surprisingly, activity was dependent on heterodimerisation with STAT1 activated by IL-4 and Oncostatin M. Our study paves the way to preclinical stages of a clinical trial with hMSC, and suggests a non-canonical JAK-STAT signaling of unphosphorylated STAT3 in immunomodulatory effects of hMSCs.

Diamond‐Blackfan anaemia: three patterns of in vitro response to haemopoietic growth factors

Culture of bone marrow from patients with Diamond‐Blackfan anaemia (DBA) has previously shown a variable progenitor response to growth factor stimulation. An extensive standardized study has now been undertaken to investigate the presence of distinct sub‐groups in this disorder. In vitro response of bone marrow progenitors to recombinant human growth factors, including stem cell factor, was examined in 18 DBA patients and five normal donors, assessing BFU‐E, CFU‐GM and CFU‐GEMM development. In 16 of the DBA patients a synergistic response to combinations of growth factors was observed with optimal growth in cultures containing erythropoietin, interleukin‐3 and stem cell factor. Growth factor induced erythroid response formed three distinct groups, based on BFU‐E numbers: type I (mean age 4.87 years) showed > 70% normal erythroid response; type II (mean age 13.87 years) showed < 70% normal; and type III (mean age 15.29 years) < 5% normal. CFU‐GM response also followed the trigrouping. The results suggest more than one pathogenic mechanism for the erythroid failure in DBA, indicating DBA may be composed of more than one distinct disorder, and further suggest the defect in DBA may not be confined to the erythroid series.

Colocalization analysis of sialomucins CD34 and CD164.

Flow cytometric protocols are employed to identify and characterize hemopoietic stem/progenitor populations before transplantation. Cell surface antigens, including CD34, are employed in this process and widely used in harvest protocols, which largely ignores the potential functional role of such antigens. Transmembrane glycoprotein sialomucins, including CD34 and CD164, have been implicated in cell‐to‐cell interactions and activation. CD164, also expressed on early hemopoietic populations, was reported to have a possible function facilitating CD34+ cells to adhere to bone marrow stroma. In this study, we employed high‐definition laser‐scanning confocal microscopy to investigate CD34 and CD164 surface co‐localization patterns on bone marrow and cord blood cells and to compare the expression patterns using a three‐dimensional computer‐generated method developed in house. Differential interference microscopy analysis revealed bone marrow membrane activity was higher than the corresponding cord blood counterpart, perhaps indicating the marrow microenvironmental nature. Fluorescence analysis of CD34 and CD164 antigens showed both were expressed first in a halo‐like pattern and second in antigen‐dense pockets. Three‐dimensional computer analyses further revealed that this pocketing corresponded to dense crest‐like surface structures appearing to rise from the point of adherence on the slide. Further, it was found that CD34 and CD164 display strong colocalization patterns on cells expressing both antigens. The dual nature of the CD34 and CD164 antigens discovered here lends further evidence to the previous literature implicating a strong functional link between these two sialomucins, which should be considered in the transplantation arena and in the function of such sialomucins as negative regulators of cell proliferation.