Preimplantation Genetic Diagnosis and Establishment of Human Embryonic Stem Cell Lines

As described in Chapter 4, PGD provides an exciting possibility for obtaining HLA-matched stem cells for treatment of siblings with bone marrow disorders. This involves preselection of HLA-matched embryos in couples with affected siblings requiring compatible stem cell transplantation, obviating the need for therapeutic cloning, which is highly controversial at the present time. However, in emergency situations, it maybe too late to wait until the HLA-matched child is born, so other possible approaches need to be developed. For example, some of the tested embryos fail to reach the developmental stage to be considered for transfer, so they might be used for establishment of embryonic stem (ES) cell lines. Alternatively, partially matched embryos may be also considered for this purpose, provided the patients provide consent.

So PGD, in addition to improving access to HLA-compatible stem cell transplantation also provides a novel source for ES cells [1,2]. Although ES cells are usually derived from culture of the inner cell mass of the preimplantation blastocyst, as shown in mice and in humans [3, 4], possibility of the establishment of ES cells from morula in mink and cow, and also from embryonic germ cells was also reported [5-7]. The current NIH repository of ES cells contains 78 lines, of which only 11 have met NIH scientific criteria (see NIH website), including the presence of L-alkaline phosphatase (TRA-2-39), Oct-4, telomerase, high-molecular-weight glycoproteins (antibodies TRA-1-60, TRA-1-81), stage-specific embryonic antigens (SSEA-3,

SSEA-4), euploid karyotype, and teratoma formation in SCID mice [8].

We developed an original technique for the establishment of ES cell lines from human embryos at the morula stage, which were shown to meet all the NIH criteria. Similar to the ES cells established from human blastocysts, the morula-derived ES cells were obtained from embryos donated by patients who gave an informed consent [9]. For establishing human ES cell lines from the morula-stage embryos, zona pellucida is removed and morula placed under middle-density feeder layer. Within several days, cells outgrow and spread into the feeder layer. The primary cell disaggrega-tion is performed with EDTA, and the loose cells are transferred back to the feeder layer to proliferate. Fast proliferating colonies are isolated and propagated further. The typical human morula-derived stem cells are shown in Figure 7.1.

In contrast to ES cell line derivation from morula, the establishment of the human ES cells from blastocyst involves immunosurgery, requiring the isolation and placement of the inner cell mass (ICM) on a feeder layer. The primary cell disaggregation is performed with EDTA and the rounded cells are transferred back to a fresh feeder. No morphological differences between human ES cells originating from ICM and from morula were observed, except for the morula-derived ES cells being more heterogeneous (see Figure 7.1) as well as the pattern of above marker expression (Table 7.1). The established human ES cell lines

0 0

Post a comment