Long-term in vivo reconstitution of hematopoiesis by human umbilical cord blood-derived highly immat...

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" Long-term in vivo reconstitution of hematopoiesis by human umbilical cord blood-derived highly immature stem cells "
N.-U.-I. Hashmi R. E. Morris

Document Type	:	Latin Dissertation
Language of Document	:	English
Record Number	:	1113087
Doc. No	:	TLpq304364303
Main Entry	:	N.-U.-I. Hashmi
	:	R. E. Morris
Title & Author	:	Long-term in vivo reconstitution of hematopoiesis by human umbilical cord blood-derived highly immature stem cells\ N.-U.-I. HashmiR. E. Morris
College	:	University of Cincinnati
Date	:	1997
student score	:	1997
Degree	:	Ph.D.
Page No	:	227
Abstract	:	Three Hematopoietic Stem Cell (HSCs) enriched fractions (i.e. CD34, CD34Lin and CD34LinRh were isolated using immunoaffinity columns and/or Fluorescence Activated Cell Sorter (FACS). The stem cell potential of the isolated cell fractions was substantiated by the short term Methylcellulose in vitro assay. The fractions were tail vein injected into sublethally irradiated Severe Combined Immune-Deficient (SCID) mice and the major hematopoietic organs of the mice were evaluated at different time points for engraftment and multilineage differentiation. High levels of multilineage engraftment were obtained as assessed by different techniques of cell and molecular biology including polymerase chain reaction (PCR) for human beta-globin gene, in situ hybridization for human specific Alu-1 sequences, human specific CD45 flow cytometry and isoelectric focusing (IEF) gel electrophoresis. No exogenous human cytokines were given to the SCID mice suggesting that the Human Umbilical Cord Blood (HUCB) derived cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Differentiation into erythroid, myeloid and megakaryocytic lineages was also demonstrated by isoelectric focusing (IEF) gel electrophoresis and in situ hybridization for human specific Alu-1 sequences. The engraftment was quantitated by the in situ hybridization technique and the human specific CD45 flow cytometry. Quantitatively significant engraftment could be maintained for long terms (up to week thirty) in the injected animals providing evidence for the multipotency of the HUCB derived highly immature cells to reconstitute hematopoiesis in the SCID mice. In contrast to the previous and concurrent studies where only unseparated or mononuclear cell populations were shown to engraft in the SCID mice, this study provides data on multilineage engraftment of the most immature HUCB derived lineage negative and Rhodamine123-dull cell populations. Besides the long term engraftment as proof for a self renewing cell population in the isolates, serial transplantation of cells from one engrafted SCID mouse to another was also carried out up to the third generation of mice. In the serial transplantation group, positive human engraftment was observed at week 32 in the SCID mice. In a small number of animals, cells infected with a recombinant adeno-associated viral (rAAV) vector containing Fanconi anemia group-C (FACC) gene linked to a neomycinResistant (Neo gene were injected. Using NeoR-specific primers, PCR analysis of the genomic DNA from organs of the SCID mice was performed to assess engraftment of the infected cells. In this group, about half of the animals showed the presence of the neomycin resistant gene. Ultrastructural analysis of the stem cell fractions was also performed but no conclusive results could be obtained. The data from these experiments suggest that the enriched fractions (both non-manipulated and genetically manipulated) from HUCB contain HSCs with properties of engraftment, differentiation and self renewal. This in vivo data on hematopoiesis by the HUCB derived highly immature stem cell fractions will be useful to gain new insights into the biology of hematopoiesis and to evaluate the gene transfer potential of the cells for a variety of clinical disorders.
Subject	:	Biological sciences
	:	Cellular biology
	:	Cellular biology
	:	Molecular biology